The population genetic structure of Japanese Spanish mackerel (Scomberomorus niphonius) in the East China Sea and Yellow Sea was analyzed using amplified fragment length polymorphism (AFLP). A total of 247 putative loci were detected by four primer combinations among 83 individuals of Japanese Spanish mackerel collected from Ganyu, Ningbo, Wenzhou, Cheju Island and Nagasaki, 129 of which were polymorphic (52.23%). The proportion of polymorphic loci and Nei’s genetic diversity for five populations ranged from 38.54% (Ganyu) to 45.70% (Nagasaki), and from 0.0808 (Cheju Island) to 0.0984 (Nagasaki). AMOVA analysis and pairwise FST revealed significant genetic differentiation among five populations. The UPGMA tree revealed the significant geographic structure in this species. Considering the high hydrological connectivity of this region and the species pelagic life history, retention of larvae, different migration route and different spawning season may be responsible for the significant genetic differentiation among populations in the East China Sea and Yellow Sea.