During the 1940s and 1950s, McClintock discovered transposition and used it to show how genes are responsible for turning physical characteristics on or off. She developed theories to explain the repression or expression of genetic information from one generation of maize plants to the next. Encountering skepticism of her research and its implications, she stopped publishing her data in 1953. Later, she made an extensive study of the cytogenetics and ethnobotany of maize races from South America. McClintock's research became well understood in the 1960s and 1970s, as researchers demonstrated the mechanisms of genetic change and genetic regulation that she had demonstrated in her maize research in the 1940s and 1950s. Awards and recognition for her contributions to the field followed, including the Nobel Prize for Physiology or Medicine, awarded to her in 1983 for the discovery of genetic transposition; she is the only woman to receive an unshared Nobel Prize in that category.

Barbara McClintock was born in Hartford, Connecticut, the third of four children of physician Thomas Henry McClintock and Sara Handy McClintock. She was independent from a very young age, a trait McClintock described as her "capacity to be alone". From about the age of three until the time she started school, McClintock lived with an aunt and uncle in Brooklyn, New York in order to reduce the financial burden on her parents while her father established his medical practice. She was described as a solitary and independent child, and a tomboy. She was close to her father, but had a difficult relationship with her mother.

McClintock completed her secondary education at Erasmus Hall High School in Brooklyn. She discovered science at high school, and wanted to attend Cornell University to continue her studies. Her mother resisted the idea of higher education for her daughters, believing it would make them unmarriageable. The family also had financial problems. Barbara was almost prevented from starting college, but her father intervened, and she entered Cornell in 1919.

McClintock began her studies at Cornell's College of Agriculture in 1919. She studied botany, receiving a BSc in 1923. Her interest in genetics had been sparked when she took her first course in that field in 1921. The course was based on a similar one offered at Harvard University, and was taught by C. B. Hutchison, a plant breeder and geneticist. Hutchinson was impressed by McClintock's interest, and telephoned to invite her to participate in the graduate genetics course at Cornell in 1922. McClintock pointed to Hutchinson's invitation as the reason she continued in genetics: "Obviously, this telephone call cast the die for my future. I remained with genetics thereafter." Although it has been reported that women could not major in genetics at Cornell, and therefore her MA and PhD— earned in 1925 and 1927, respectively—were officially awarded in botany, recent research has revealed that women did earn graduate degrees in Cornell's Plant Breeding Department during the time that McClintock was a student at Cornell.

During her graduate studies and postgraduate appointment as a botany instructor, McClintock was instrumental in assembling a group that studied the new field of cytogenetics in maize. This group brought together plant breeders and cytologists, and included, Charles R. Burnham, Marcus Rhoades, George Beadle (who became a Nobel laureate in 1958 for showing that genes control metabolism), and Harriet Creighton. Rollins Adams Emerson, head of the Plant Breeding Department, supported these efforts, although he was not a cytologist himself. McClintock's cytogenetic research focused on developing ways to visualize and characterize maize chromosomes. This particular part of her work influenced a generation of students, as it was included in most textbooks. She also developed a technique using carmine staining to visualize maize chromosomes, and showed for the first time the morphology of the 10 maize chromosomes. By studying the morphology of the chromosomes, McClintock was able to link, to a specific chromosome, groups of traits that were inherited together. Marcus Rhoades noted that McClintock's 1929 Genetics paper on the characterization of triploid maize chromosomes triggered scientific interest in maize cytogenetics, and attributed to his female colleague 10 of the 17 significant advances in the field that were made by Cornell scientists between 1929 and 1935.

In 1930, McClintock was the first person to describe the cross-shaped interaction of homologous chromosomes during meiosis. During 1931, McClintock and a graduate student, Harriet Creighton, proved the link between chromosomal crossover during meiosis and the recombination of genetic traits. They observed how the recombination of chromosomes and the resulting phenotype formed the inheritance of a new trait. Until this point, it had only been hypothesized that genetic recombination could occur during meiosis, although it had been shown genetically. McClintock published the first genetic map for maize in 1931, showing the order of three genes on maize chromosome 9. This information provided necessary data for the crossing over study she published with Creighton. In 1938, she produced a cytogenetic analysis of the centromere, describing the organization and function of the centromere.

McClintock's breakthrough publications, and support from her colleagues, led to her being awarded several postdoctoral fellowships from the National Research Council. This funding allowed her to continue to study genetics at Cornell, the University of Missouri, and the California Institute of Technology, where she worked with E. G. Anderson. During the summers of 1931 and 1932, she worked at Missouri with geneticist Lewis Stadler, who introduced her to the use of X-rays as a mutagen. (Exposure to X-rays can increase the rate of mutation above the natural background level, making it a powerful research tool for genetics.) Through her work with X-ray-mutagenized maize, she identified ring chromosomes, which form when the ends of a single chromosome fuse together after radiation damage. From this evidence, McClintock hypothesized that there must be a structure on the chromosome tip that would normally ensure stability. She showed that the loss of ring-chromosomes at meiosis caused variegation in maize foliage in generations subsequent to irradiation resulting from chromosomal deletion. During this period, she demonstrated the presence of what she called the nucleolar organizers on a region on maize chromosome 6, which is required for the assembly of the nucleolus.[citation needed]

McClintock received a fellowship from the Guggenheim Foundation that made possible six months of training in Germany during 1933 and 1934. She had planned to work with Curt Stern, who had demonstrated crossing-over in Drosophila just weeks after McClintock and Creighton had done so; however, in the meantime, Stern emigrated to the United States. Instead, she worked in Germany with geneticist Richard B. Goldschmidt. She left Germany early, amid mounting political tension in Europe, and returned to Cornell, remaining there until 1936, when she accepted an Assistant Professorship offered to her by Lewis Stadler in the Department of Botany at the University of Missouri–Columbia.

During her time at Missouri, McClintock expanded her research on the effect of X-rays on maize cytogenetics. McClintock observed the breakage and fusion of chromosomes in irradiated maize cells. She was also able to show that, in some plants, spontaneous chromosome breakage occurred in the cells of the endosperm. Over the course of mitosis, she observed that the ends of broken chromatids were rejoined after the chromosome replication. In the anaphase of mitosis, the broken chromosomes formed a chromatid bridge, which was broken when the chromatids moved towards the cell poles. The broken ends were rejoined in the interphase of the next mitosis, and the cycle was repeated, causing massive mutation, which she could detect as variegation in the endosperm. This cycle of breakage, fusion, and bridge, also described as the breakage–rejoining–bridge cycle, was a key cytogenetic discovery for several reasons.[citation needed] First it showed that the rejoining of chromosomes was not a random event, and secondly it demonstrated a source of large-scale mutation. For this reason, it remains an area of interest in cancer research today.

Although her research was progressing at Missouri, McClintock was not satisfied with her position at the University. She recalled being excluded from faculty meetings, and was not made aware of positions available at other institutions. In 1940 she wrote to Charles Burnham, "I have decided that I must look for another job. As far as I can make out, there is nothing more for me here. I am an assistant professor at $3,000 and I feel sure that that is the limit for me." Initially, McClintock's position had been especially created for her by Stadler and may have depended on his presence. McClintock believed she would not gain tenure at Missouri, although according to some accounts she knew she would be offered a promotion by Missouri in the spring of 1942. Recent evidence reveals that McClintock more likely decided to leave Missouri because she had lost trust in her employer and in the University administration. In early 1941 she was invited by the Director of the Department of Genetics at Cold Spring Harbor to spend her summer there. She took a leave of absence from Missouri in hopes of finding a position elsewhere. She also accepted a visiting Professorship at Columbia University, where her former Cornell colleague Marcus Rhoades was a professor. He offered to share his research field at Cold Spring Harbor on Long Island.[clarification needed] In December 1941 she was offered a research position by Milislav Demerec, the newly appointed acting director, and she joined the staff of the Carnegie Institution of Washington's Department of Genetics Cold Spring Harbor Laboratory.