The obtuse barracuda (Sphyraena obtusata) is a pelagic–neritic species that usually swims at or near the water surface. To investigate the genetic diversity of S. obtusata populations using the Amplified Fragment Length Polymorphism (AFLP) marker, 26 specimens were collected from Bahrakan, Dayyer, and Bandar Abbas. Genomic DNA was extracted from dorsal fin tissue using the phenol–chloroform method. Five MseI/EcoRI primer combinations were used. Data analysis showed that the mean percentage of polymorphic loci among the three populations was 76.10%, with an average genetic diversity of 0.289 ± 0.022 and an average Shannon’s index of 0.432 ± 0.031, indicating notable genetic diversity within the populations. Analysis of Molecular Variance (AMOVA) revealed that 16% of the variation occurred among populations and 84% within populations at a 99% confidence level. The mean gene flow among populations was 2.37, suggesting effective migration between populations due to the species’ pelagic eggs and larvae. The genetic distance dendrogram indicated that population differentiation corresponded to their geographical separation.