The heat shock proteins of the virilis group of Drosophila are analyzed by one- and two-dimensional polyacrylamide gel analysis. This group consists of the two closely related but distinct virilis and montana phylads. The analysis reveals that some of the heat shock proteins are highly conserved among the two phylads while others are not. The 83-, 72-, and 69-kdalton proteins comigrate in all species examined. There is, however, a noticeable trend toward greater molecular weight variability in the smaller heat shock proteins. In general, the heat shock protein patterns within each phylad follow the proposed phylogenetic relationships with some exceptions. D. ezoana and D. littoralis, both members of the montana phylad, exhibit heat shock protein patterns more similar to those of the virilis phylad. The data also demonstrate that the montana phylad has almost two times the heat shock allele members that the virilis phylad has. It is also shown that F1 and F2 hybrid flies of crosses between Drosophila species having different patterns of heat shock proteins show Mendelian segregation of alleles. After several generations of inbred growth, however, the pattern of heat shock protein synthesis in reciprocal hybrids each resembles that of the paternal parent. The implications of these findings are discussed.