Theophilus Shickel Painter studied the structure and function of chromosomes in the US during in the early to mid-twentieth century. Painter worked at the University of Texas at Austin in Austin, Texas. In the 1920s and 1930s, Painter studied the chromosomes of the salivary gland giant chromosomes of the fruit fly (Drosophila melanogaster), with Hermann J. Muller. Muller and Painter studied the ability of X-rays to cause changes in the chromosomes of fruit flies. Painter also studied chromosomes in mammals. He investigated the development of the male gamete, a process called spermatogenesis, in several invertebrates and vertebrates, including mammals. In addition, Painter studied the role the Y-chromosome plays in the determination and development of the male embryo. Painter's research concluded that egg cells fertilized by sperm cell bearing an X-chromosome resulted in a female embryo, whereas egg cells fertilized by a sperm cell carrying a Y-chromosome resulted in a male embryo. Painter's work with chromosomes helped other researchers determine that X- and Y-chromosomes are responsible for sex determination.

The Y-chromosome is one of a pair of chromosomes that determine the genetic sex of individuals in mammals, some insects, and some plants. In the nineteenth and twentieth centuries, the development of new microscopic and molecular techniques, including DNA sequencing, enabled scientists to confirm the hypothesis that chromosomes determine the sex of developing organisms. In an adult organism, the genes on the Y-chromosome help produce the male gamete, the sperm cell. Beginning in the 1980s, many studies of human populations used the Y-chromosome gene sequences to trace paternal lineages. In mammals, the Y-chromosomes contain the master-switch gene for sex determination, called the sex-determining region Y, or the SRY gene in humans. In most normal cases, if a fertilized egg cell, called a zygote, has the SRY gene, the zygote develops into an embryos that has male sex traits. If the zygote lacks the SRY gene or if the SRY gene is defective, the zygote develops into an embryo that has female sex traits.