Phylogeographic analysis of the bitterling Tanakia limbata in western Japan was conducted to identify the origin of the fish population in Ehime, Shikoku Island. A survey of rivers and spring-fed ponds on the Matsuyama Plain, Ehime, indicated that T. limbata had become distributed on the plain over a period of 19 years to the present day. Sequences of the mitochondrial cytochrome b gene indicated that the 42 individuals sampled included 4 haplotypes, which were shared with a T. limbata population in the Yabe River, Fukuoka, Kyushu Island. All 4 haplotypes belonged to the West Kyushu group of T. limbata. On the other hand, T. limbata comprising the West Seto clade inhabits western Honshu and eastern Kyushu, which regions shared the same paleoriver system with Ehime 20,000 years BP. These results suggest that individuals of T. limbata presently collected in Ehime originated from the Yabe River or adjacent waters, having artificially transported over the mountain chain that may act as a natural barrier separating the West Kyushu and West Seto groups. To conserve an endangered native bitterling, T. lanceolata, in Ehime, management of the introduced T. limbata, so as to prevent competition and hybridization with the former, is necessary.