The genetic relationships among the five species of the subgenus Hitrodrosophila, d. sexvittata, D. trilineata, D. alboralis, D. histrioides and D. nokogiri were investigated by isozyme and soluble proteins. The 15 loci were detected electrophoretically in the 10 isozymes. The five loci, Aph-1, α-Gpdh, Mdh-2, Odh, and Sdh were monomorphic in all species, and specially Mdh-2 was same allele. As for the genetic relationship based on the results from the isozyme analysis, D. sexvittata and D. trilineata were found to have the most close genetic relationship (S=0.634), and the relationship between D. nokogiri and D. trilineata showed the farthest (S=0.307). The average genetic similarity among the five species was found to be 0.818. The soluble protein patterns of D. sexvittata and D. trilineata by SDS polyacrylamide gel electrophoresis were similar. The difference of protein pattern among the other 3 species were confirmed. The genetic distance of five species was calculated by means of two-dimensional gel electrophoresis. The genetic distance between D. sexvittata and D. trilineata was 0.1139, the lowest of all, between D. trilineata and D. histrioides, 0.1883, between D. nokogiri and D.sexvittata, 0.4978, the highest of all. The dendrograms established by isozyme and soluble protein analysis had same patterns. Clustered patterns as follows; first, D. sexvittata and d. trilineata were clustered, and D. histrioides, D. alboralis and D. nokogiri followed the first one in order.