Information on the genetic structure of fish species is essential for optimizing fisheries management and stock improvement programs. Ten microsatellite loci were analyzed to study the genetic variation in six populations of the cobia (Rachycentron canadum). Seven of the ten loci analyzed were polymorphic in all the populations. Locus Rca 1B-H09 had the highest numbers of alleles (18), while the locus Rca 1B-A10 and Rca 1B-E08A had the lowest (14). All the studied populations deviated from Hardy–Weinberg equilibrium proportions at a number of loci, mostly due to the deficiency of heterozygosities. A moderate level of population differentiation (F ) was observed among populations; however, highest significant differentiation was between the Dayer and Pozm populations. The genetic distance computed by Nei between the Dayer and Beris populations was higher than the genetic distances between all other population pairs. The study revealed a relatively moderate level of genetic variation at microsatellite loci within and between cobia populations