The biological indicator should be developed in the ecoregion where biota is relatively identical. Furthermore, as stream biota widely varies from headwater to downstream, it is necessary to define segment based ecoregions where biota is longitudinally identical. In this study, we delineated segment based ecoregions for ten rivers belonging to the northwest Kyushu ecoregion based on the degree of similarity of the fish fauna. The northwest Kyushu ecoregion was divided into five segment based ecoregions. As a result, fish fauna was more clearly characterized by the differences in community structure between segment based ecoregions than those between watersheds. Moreover, distinguishing segment based ecoregions, enabled us to compare the integrity of fish fauna between watersheds, which had been previously difficult to examine.