Over thirty-eight species of Gossypium are recognized. Six species are tetraploid, including the most widely cultivated species, G. barbadense and G. hirsutum. The genus contains very diverse germplasm. We have been developing methods to work with cotton at the cellular and molecular level to fully utilize this germplasm. We have greatly improved the technique for cotton chloroplast DNA isolation. This modified procedure eliminates the need for costly and time consuming cesium chloride-ethidium bromide density gradients and provides chloroplast DNA significantly less contaminated with nuclear DNA. We have been using this technique to map plastid DNA variation in Gossypium. Although chloroplast DNA in is very highly conserved, species and races of Gossypium which are morphologically very similar can be differentiated, e.g., G. arboreum can be distinguished from G. barbadense, G. darwinnii, and two races of G. hirsutum with the restriction endonuclease BamHI. G. arboreum also has banding patterns different from a third race of G. hirsutum, G. lanceolatum, and G. mustellinum with SalI. Thus, using two different enzymes, G. arboreum can be distinguished from 5 other species of Gossypium. In addition, we have been using plastid DNA variation to help clarify questions of evolution and development in Gossypium. During the development of modern cultivated cotton, allotetraploid species were formed from diploid species. Whether the six tetraploid species developed from one ancestral cross, or several different crosses is the subject of debate.