The genetic relationships among the five species of the subgenus Hirtodrosophila, D. sexvittata, D. trilineata, D. alboralis, D. histrioides and D. nokogiri were investigated by numerical analysis, isozyme and soluble proteins. The numerical analysis according to the 17 character was performed by cluster analysis. The genetic similarity coefficient (Gower`s) between D. sexvittata and D. trilineata was 0.894, the highest of all, between D. histrioides and D. trilineata, 0.643, between D. alboralis and D. histrioides, 0.571, and between D. nokogiri and D. alboralis, 0.259, the lowest of all. The isozyme analysis was performed to establish the genetic distance (Nei, 1972) and genetic similarity (Rogers, 1972). The 15 loci were detected electrophoretically in the 10 isozymes. The five loci, Aph-1, α-Gpdh, Mdhm, Odh, and Sdh were monomorphic in all species, and specially Mdhm 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; D=0.358), and the relationship between D. nokogiri and D. trilineata showed the reverse (S=0.634; D=1.132). The average genetic distance and genetic similarity among the five species was found to be 0.438 and 0.818, respectively. The soluble protein patterns of D. sexvittata and D. trilineata by SDS polyacrylamide gel electrophoresis appeared similar. The differences 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 numerical analysis, isozyme analysis 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.