Polymorphism of 12 microsatellite markers was analyzed to investigate genetic variability and structure in broodstock populations of the Mediterranean sea bass Dicentrarchus labrax. Two broodstocks of wild origin (AN, EN) that had been collected from the different locations of the Aegean Sea, as well as one of culture origin (after three generations in captivity) (AF) were used in the analysis. In total, 319 individuals from these three sample groups were scored. The average allele numbers of the 12 microsatellite loci were 9.16, 8.9, and 11.5 for AF, AN and EN groups, respectively. Wild groups showed a higher variation than the cultured strains, and significant deviations from Hardy-Weinberg Equilibrium were observed. The observed heterozigosity values for AF, AN and EN groups were 0.502, 0.608 and 0.620, respectively. The significant linkage equilibrium was found for all groups between various loci. Furthermore, AMOVA results confirmed a significant genetic differentiation between all populations with an estimated F-ST value of 0.113 (P<0.001), in accordance with the pair-wise estimates between groups. The results showed that genetic variability was the lowest for the culture-originating broodstock population, possibly due to the inbreeding effect. Bayesian analysis showed that individuals in populations represented 5 genetic groups (K=5). However, two wild broodstock populations also showed a low genetic variability, indicating the importance of collecting fish from different geographic locations while establishing any broodstock population in a hatchery.