We investigated the genetic structure of Korean and Japanese ayu Plecoglossus altivelis populations by examining 669 individuals from 14 populations using three microsatellite loci. Genetic variation did not differ significantly among the populations examined in terms of allelic number and heterozygosity. Korean populations were genetically close to each other, implying that persistent gene flow has occurred in these populations. This suggests that eastern populations in Korea form a single large population and all of the Korean populations are distinct from the Japanese populations. Pairwise population $F_{ST}$ estimates, principal component analyses, and a neighbor-joining tree showed that genetic separation between the southern and pooled eastern coast populations was probably influenced by restricted gene flow. Hierarchical analysis of molecular variance (AMOVA) revealed a weak but significant genetic structure among three ayu groups (eastern and southern coasts of Korea and the Japan coast), and no genetic variation within groups. The estimated genetic population structure and potential applications of microsatellite markers may aid in the proper management of ayu populations.