Although many fish passages have been constructed to facilitate fish migration through weirs, it is difficult to determine their effectiveness, especially for uncommon species. In this study, molecular methods were used to evaluate fish migration in the Ota River in Hiroshima, Japan. Study sites included a 55-km reach with nine weirs and a 10-km reach of a tributary that had three dams. Dams on the branch of the Ota River were built in 1959, 1961, and 2001, and none allowed the passage of fish. Fin samples from four fish species, Nipponocypris temminckii, Rhinogobius flumineus, Liobagrus reini and Pseudogobio esocinus, were collected over a two-year period for DNA extraction. Amplified fragment length polymorphisms (AFLPs) or short tandem repeat method (STR) were used for analysis. Few differences were found throughout the Ota River in both upstream and downstream regions. Over the two years of analysis, it was assumed that neither Nipponocypris temminckii nor Rhinogobius flumineus had divided with the downstream site; however, several weirs seemed to be impeding the migration of these species. Liobagrus reini, which are the weakest swimmer of the four species, showed less effect. We consider that these results are due to both their swimming performance and primary range of activity. Also, several weirs may have impacted the divided populations in the short term, though the populations could easily recover. The swimming performance, local population range and local population size of each species are important factors when considering effective designs for restoring fish migration.