Black pomfret Parastromateus niger is a commercially important fishery resource in the Persian Gulf but harvesting its stocks lacks genetic identification of populations. AFLP technique was applied to analyze genetic diversity and population structure of 32 fish from coastal waters of Bandar Abbas, Bushehr and Abadan with 7 EcoRI/MseI primer pair combinations. In total, 381 bands were produced of which, 46 were polymorphic (12.07%). Percentage of polymorphic bands was higher in Bushehr samples (91.30%) than in Abadan (84.78%) and Bandar Abbas (73.91%) samples. The highest level of heterozygosity based on Nei’s coefficient and Shannon’s index was observed in Bushehr fish (0.38±0.16 and 0.54±0.21). Observed and effective alleles ranged from 1.73±0.44 and 1.53±0.40 in Bandar Abbas samples to 1.91±0.28 and 1.70±0.34 in Bushehr samples. The average F_st was 0.19 indicating high genetic differentiation among the three locations. Gene flow with mean of 1.93 was the lowest level between Bandar Abbas and Abadan (1.24). Nei's genetic identity revealed the least genetic similarity between the samples of Bandar Abbas and Abadan (0.77). AMOVA analysis demonstrated 81% of the genetic variation within populations and 19% among populations. The UPGMA dendrogram clustered all 32 individuals into 3 groups. In some cases individuals from the same region were grouped together but in most cases, gene exchange was observed to be common among the groups. Analyses provided evidence for genetic differentiation among the three locations, indicating separate populations of black pomfret in the northern Persian Gulf.