Ayu (Plecoglossus altivelis altivelis), an important freshwater fisheries resource and popular recreational fishing species, lives for only one year and has a single breeding season. To supplement increased recreational fishing demand, huge numbers of wild-born landlocked juvenile Ayu have been translocated from Lake Biwa into most Japanese rivers for more than 50 generations. Hatchery-born amphidromous fish (including amphidromous and landlocked form hybrids) have also been released for many generations.Hatchery-born fish have low survival and maladapted behaviour in the wild. Landlocked and amphidromous forms of Ayu easily hybridise, but survival of progeny of landlocked forms is very low in seawater. Repeat backcrossing may cause introgression of landlocked forms into amphidromous populations, but this has not been previously identified.Study objectives using genetic data from Ayu from 118 locations throughout the distribution of this species in Japan are to describe contemporary population structure, genetic diversity, and admixture proportions of Ayu forms in populations, and to evaluate how human-induced translocation has affected population genetic structure.The analyses of published genotypes of 12 microsatellite markers provide strong evidence for very high gene flow between populations, but population structure has been retained in several regions, and several populations are nested. Genetic diversity is surprisingly homogeneous. Hybridisation between landlocked and amphidromous forms has occurred in all populations, with a mean hybrid proportion (± standard deviation) of 37 ± 10%, ranging 15%–60%.Results are discussed in relation to the conservation and management of this species. Recommendations are made to reduce translocation and hatchery releases, by establishing rivers and/or areas in every prefecture where translocation does not occur. Release of juveniles is of value for short-term management objectives, but management of spawning escapements, and improving the spawning and nursery habitat are important for this species’ long-term sustainability.