In the early 20th century, the groundbreaking work on the genetics of Drosophila, the fruit fly, by such notable scientists as Thomas Hunt Morgan and Calvin Bridges, and on the genetics of maize (corn) by Edward Murray East, Donald F. Jones, and Rollins A. Emerson helped to establish genetics as the most forward-looking area of biological science. Corn genetics had its own particular interest, as corn was at the center of a large agricultural industry, serving as food, animal fodder, and as a source of engine fuel and distilled liquor. Beyond these economic incentives, the corn plant is a useful experimental tool as it has easily observable traits, and each kernel is the result of a separate genetic cross.

McClintock's focus at Cornell, from her undergraduate days through her Ph.D. and postdoctoral work, was on the cytogenetics of maize, that is, bringing together classic genetics and the study of maize cells. After earning her Ph.D. at Cornell in 1927, McClintock stayed on as an instructor until 1931. As part of a cohort of Cornell graduates who worked with Rollins Emerson, which included George Beadle, Charles Burnham, and Marcus Rhoades, McClintock founded the discipline of maize cytogenetics.

During this period at Cornell, McClintock collaborated with graduate student Harriet Creighton on a series of experiments that demonstrated that a genetic phenomenon called "crossing-over" had a cellular explanation. In most organisms, chromosomes are paired, with one chromosome of each pair being inherited from each parent. It had long been suspected that, during the process of cellular division called meiosis, parts of one chromosome inherited from one parent could change place with their analogous parts on the chromosome from the other parent. McClintock and Creighton were able to link a genetic trait with a part of a chromosome observable under a microscope and thus demonstrate the physical basis of genetic crossing-over. McClintock knew she had made a significant breakthrough. As she wrote to Charles Burnham in November 1929, "I haven't solved the situation totally but I have opened up some new things which are very valuable, cytologically, & I sincerely think I can solve a lot more as it is turning out more interesting than I had anticipated even; at least I feel more confidence than I did 10 days ago when I felt I had hopelessly failed to solve anything." McClintock and Creighton's paper, published in 1931 in the Proceedings of the National Academy of Sciences, became a classic in the field.