Utilizing the technique of DNA – DNA hybridization, we have characterized the degree of genetic variability in single-copy DNA both within and between several species of Drosophila. The results of intraspecific variation studies indicate considerable variation both for levels of nucleotide heterozygosity (estimated to be over 2%) as well as for insertions–deletions. Interspecific studies confirm this great deal of variability and further establish an extreme heterogeneity within Drosophila genomes for rates of divergence. This heterogeneity is much more extreme than that seen between exons and introns. The degree of single-copy DNA divergence generally supports phylogenetic affinities deduced from more traditional methods. However, exceptions occur where single-copy DNA divergence is not correlated with other properties such as degree of chromosomal differentiation, morphology, or ability to form interspecific hybrids. We argue that single-copy DNA divergence as measured by DNA–DNA hybridization is an accurate indicator of phylogenetic relationships and therefore sheds light on the evolution of other biological properties. Many, if not most, evolutionary tests require an accurate phylogeny of the group being studied and DNA, because of the high information content inherent within the molecule, offers the best hope of deriving true phylogenies.Key words: DNA evolution, Drosophila, molecular evolution, phylogenetics, DNA–DNA hybridization.