We used a before–after control–impact design to quantify the genetic effects of the large piscivorous Japanese Spanish mackerel (Scomberomorus niphonius) stock enhancement program on wild populations in the Seto Inland Sea. Samples of 1424 wild and 230 hatchery fish collected from 13 sites around Japan were genotyped using five microsatellite markers. A total of 758 wild and 103 hatchery fish were sequenced for the mitochondrial DNA D-loop region. The population structure of Japanese Spanish mackerel was panmictic around Japan. Hatchery fish had significantly lower genetic diversity indices than did wild fish. However, there was no significant change in any of the diversity indices in the Seto Inland Sea, despite the substantial genetic mixing proportion of hatchery-origin genes (7.8%–14.5% from releases in 2001 and 2002), a conclusion supported by simulations. The estimated effective population size was surprisingly small (∼430–970) but stable in the Seto Inland Sea compared with the large census size. A Ryman–Laikre effect was not likely in the Japanese Spanish mackerel.