The distributions of two bitterling fish (subfamily: Acheilognathinae), Tanakia lanceolata and T. limbata, overlap in western Japan. Acheilognathinae fish lay their eggs in the gills of freshwater bivalves, and the early juvenile stage develops in the gills. Populations of freshwater bivalves are declining worldwide, which has limited the number of spawning substrate for bitterlings. T. limbata has been artificially introduced to some rivers in Ehime, Japan, where it coexists with native T. lanceolata, and some hybrids have been observed. We collected both species from several sites in western Japan, and from the Kunichi River system in Ehime, and analyzed genetic population structure based on six microsatellite loci and sequences of the mitochondrial cytochrome b gene. Structure analysis identified three genetically distinct populations: T. lanceolata, T. limbata “West Kyushu”, and T. limbata “Setouchi”. Two clades of T. limbata were also supported by molecular phylogenetic analyses based on cytochrome b. Hybrids in Ehime originated mostly from interbreeding between male T. lanceolata and female T. limbata “West Kyushu”, and made up 10.2% of all collected fish, suggesting that hybrids occurred frequently between females of colonizing species and males of native species. On the other hand, interspecific hybrids were detected at rates of 40.0%, 20.0%, and 17.6% in the Ima River (Fukuoka), Midori River (Kumamoto), and Kase River (Saga), respectively, which are naturally sympatric regions. We found a few T. limbata “Setouchi” in the Midori and Kase Rivers, which were supposed to be introduced from other regions, coexisting with native T. limbata “West Kyushu”, and this cryptic invasion may have triggered the interspecific hybridization. These results suggest that artificial introduction of a fish species, a decline in the unionid population, and degradation of habitat have caused broad hybridization of bitterlings in western Japan.