Born in Lexington, Kentucky, Morgan studied zoology at the State College of Kentucky, graduating in 1886. He received his PhD from Johns Hopkins University in 1890 and from 1891 to 1904 was associate professor of zoology at Bryn Mawr College. He carried out his most important work between 1904 and 1928, while professor of experimental zoology at Columbia University. Here he became involved in the controversy that followed the rediscovery, in 1900, of Gregor Mendel's laws of inheritance.

Many scientists had noted that Mendel's segregation ratios fitted in well with the observed pattern of chromosome movement at meiosis. Morgan, however, continued to regard Mendel's laws with skepticism, especially the law of independent assortment, and with good reason. It was known by then that many more characters are determined genetically than there are chromosomes and therefore each chromosome must control many traits. It was also known that chromosomes are inherited as complete units, so various characters must be linked together on a single chromosome and would be expected to be inherited together.

In 1908 Morgan began breeding experiments with the fruit fly Drosophila melanogaster, which has four pairs of chromosomes. Morgan's early results with mutant types substantiated Mendel's law of segregation, but he soon found evidence of linkage through his discovery that mutant white-eyed flies are also always male. He thus formulated the only necessary amendment to Mendel's laws – that the law of independent assortment only applies to genes located on different chromosomes.

Morgan found that linkages could be broken when homologous chromosomes paired at meiosis and exchanged material in a process known as ‘crossing over’. Gene linkages are less likely to be broken when the genes are close together on the chromosome, and therefore by recording the frequency of broken linkages, the positions of genes along the chromosome can be mapped. Morgan and his colleagues produced the first chromosome maps in 1911.

For his contributions to genetics, Morgan received the Nobel Prize for physiology or medicine in 1933. A prolific writer, his most influential books – produced with colleagues at Columbia – are The Mechanism of Mendelian Heredity (1915) and The Theory of the Gene (1926).