The migratory fish ayu, Plecoglossus altivelis, has two forms: an amphidromous form found throughout the Japanese islands and a landlocked form found in Lake Biwa. Because Lake Biwa is one of the sources of the Yodo River, which flows into Osaka Bay, it is likely that there has been contact between the two forms within the Yodo River system. To examine the effects of such contact, 573 ayu were collected from the Yodo River system and several other rivers and compared through an extensive investigation of microsatellite genotyping. Multivariate and individual admixture analyses of microsatellite data showed that samples of upstream migrants collected from the Yodo River in May 2003 included many individuals assigned to the amphidromous form, although fish collections in June and July 2003 were primarily composed of individuals of the landlocked form. Samples of drifting larvae, which we considered to be derived from eggs spawned in the Yodo River system, were mostly assigned to the amphidromous form. These results imply that an indigenous population of the amphidromous form exists and reproduces in the Yodo River system. It is likely that upstream migrants of the landlocked Lake Biwa form are supplied annually as well-developed larvae drifting out from Lake Biwa, but contribute very little to the next generation.