Snails of the genus Pomacea in several regions of Japan showed genetic divergence. Phylogenetic analysis based on the mitochondrial cytochrome c oxidase subunit I gene segregated them into two major clades, one corresponding to P. insularum, the other to P. canaliculata. We developed a simple molecular method to distinguish between the two species to investigate their distribution in Japan. We identified P. canaliculata at all 16 sampling locations except Iriomote Isle, and P. insularum at four locations (Iwata, Fukuyama, Ishigaki Isle, and Iriomote Isle), supporting a previous report that P. canaliculata is the dominant species in Japan. Morphological comparison of the two species collected from the same habitat (Ishigaki Isle) revealed that most P. canaliculata had clear dark spiral bands on their shells, and that P. insularum had no or only faint spiral bands. However, it is difficult to distinguish the two species using only shell morphology.