The amount of heterochromatin in the genome of ten members of the virilis species group was determined as the length of C-band chromosome material relative to the total karyotype length. The virilis phylad (Drosophila virilis, D. novamexicana, D. americana americana, and D. americana texana) has significantly greater amounts of heterochromatin in the genome than do members of the montana phylad (D. montana, D. lacicola, D. flavomontana, D. borealis, D. ezoana, D. littoralis). Thus, the significant karyotypic change accompanying diversification of these species has involved reduction in their total constitutive heterochromatin. These changes have apparently involved reductions in the amount of centromeric heterochromatin in the autosomes.