Elucidating the scale of gene flow among populations is an important challenge for understanding the ecological dynamics and local adaptation of marine organisms. We assessed whether gene flow is restricted even at a small spatial scale in the Japanese common intertidal goby Chaenogobius annularis, using highly polymorphic DNA markers, involving the mitochondrial DNA (mtDNA) control region and 15 microsatellite DNA (msDNA), because past ecological studies have suggested low dispersal ability for rocky intertidal fishes. We found significant heterogeneities between four neighboring local populations by both mtDNA and msDNA analyses. In addition, no genetic heterogeneity was detected by either method across generations within a population; it was considered that such genetic differentiation is retained across generations and that the gene flow of this species is restricted to within a radius of a few kilometers. This is the first report showing a clear genetic subdivision in rocky intertidal fish.