The Japanese rosy bitterling, Rhodeus ocellatus kurumeus, has been affected not only by the invasion of another subspecies, R. o. ocellatus, from China, but also by habitat fragmentation. In this study, the effects of habitat fragmentation on the fitness of R. o. kurumeus were investigated. Owing to exclusion by R. o. ocellatus, R. o. kurumeus in Honshu and Shikoku has disappeared entirely, except for small populations in isolated man-made ponds in Osaka and Kagawa. In Kyushu it still occupies open water systems, into which R. o. ocellatus has only recently invaded. Meristic and genetic data show that the diversity of R. o. kurumeus is significantly lower in the isolated Osaka and Kagawa populations than the non-isolated Fukuoka population. The Osaka population is inferior to the Fukuoka population in terms of viability and growth. The viability of reciprocal inter-population hybrids between the Osaka and Fukuoka populations was, however, as high as that of the Fukuoka population. In addition to the high scores of band sharing index (BSI) in RAPD-PCR analysis, acceptance of transplanted scales among individuals, irrespective of natal pond, indicates that the Osaka population forms a highly inbred line. These results suggest that low genetic variation is associated with inbreeding depression in the small isolated Osaka populations. Consequently, the management of ponds, including the free movement of individuals, in addition to measures to prevent the invasion of R. o. ocellatus, is necessary for the conservation of R. o. kurumeus.