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Nucleotide sequences of a 0.7kb portion of the mitochondrial cytochrome b gene were analyzed using Japanese species of water striders belonging to the subgenus Macrogerris. Although the sequences were rather homogeneous among individuals within both Gerris gracilicornis and G. yezoensis, considerable variation was observed among insects showing conventionally recognized diagnostic characteristics of G. insularis. Results of molecular phylogenetic analyses clearly indicated that there are three distinct lineages, designated as types A, B, and C, among these latter insects. Comparisons of nucleotide sequences indicated that genetic distances among these lineages were much larger than those between G. gracilicornis and G. yezoensis. From PCR-RFLP analyses of insects collected from 35 localities, it was revealed that the geographic distributions of types B and C were divided between northeastern Honshu island and western Japan including Kyushu island and central to western Honshu island, whereas that of type A largely overlapped with those of the other two types. Although male adults of the three types were homogeneous in conventionally recognized diagnostic characteristics, apparent differences were observed in the shape of the head, pronotum, fore femur, and color pattern of the anterior pronotum between type A and the other two types. In addition, types B and C showed apparent dimorphism in hind wings, containing both long- and short-winged individuals, whereas type A showed only long and wide hind wings. Preliminary observations of mating behavior indicated that a precopulatory reproductive isolation is operative between type A and other two types. Based on these results, evolutionary relationships among the three lineages are discussed.