Nucleotide sequences for a 315-base-pair segment of the mitochondrial COI (cytochrome c oxidase subunit I) gene were compared among 76 individuals from 29 populations of the genus Ishizakiella. Resulting molecular phylogenetic trees consistently supported both the species assignment based on morphological data and the closest relationship between I. miurensis and I. ryukyuensis. However, these trees were equivocal as to the position of I. supralittoralis among the four ingroup species, i.e., it clustered as either sister group of I. novaezealandica or of both I. miurensis and I. ryukyuensis. The close relationship between I. miurensis and I. ryukyuensis was further supported by the fact that the two species share serration on the copulatory duct. The phylogenetic relationship, fossil record, and geographical distributions suggested that the ancestor of I. supralittoralis was the first to have separated from the common ancestor of the three Japanese species and arrived in the Japanese Archipelago before the Pleistocene. Then, the ancestor of I. miurensis was separated from that of I. ryukyuensis, with the former having migrated to the Japanese Archipelago to dominate over I. supralittoralis. The Sea of Japan populations of I. miurensis formed a paraphyletic group, suggesting that ancestors of I. miurensis migrated to the Japanese Archipelago at least twice. The formation of landbridge was perhaps important in the separation and migration of the populations. The large genetic distance between two populations of isolated islands observed for I. ryukyuensis indicated that open sea also acts as a notable barrier preventing gene flow.