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Twenty five individuals of Javelin grunter, Pomadasys kaakan, collected from Abadan (29° N and 48° E) and Bandar Abbas (27° N and 56° E) stations along the Persian Gulf, was analyzed for relationship between two populations of this species, using Amplified Fragment Length Polymorphism (AFLP). A total of 410 putative loci were detected by six primer combinations, 88 of which were polymorphic (21.46%). The proportion of polymorphic loci in the Abadan and Bandar Abbas stations was calculated 88.64 and 68.18%, respectively. Average of heterozygosity in the Abadan (0.330) was higher than Bandar Abbas (0.222), which may be correlated with the environmental and ecological conditions of these stations. Nei’s genetic distance for the two populations was estimated 0.112. The phylogenetic tree revealed a clear distinction line between the two populations, supporting that dispersal of eggs and larvae in the Persian Gulf is spatially restricted. Pattern of isolation by distance was observed in this species, indicating that the gene pool of P. kaakan in the Persian Gulf was not homogeneous. The results also showed that AFLP is a potent technique for genetic fingerprinting and species identification.