The genetic variation at 21 protein loci was investigated in six natural gilthead seabream (Sparus aurata L., 1758) populations from the Tunisian coast to analyse the amount and distribution of genetic diversity of this species. In the same way, one reared population has been studied. A strong differentiation was detected (D: 0.023-0.083, pairwise FST: - 0.0085-0.2654) between our samples, mainly attributable to the existence of two divergent groups of populations: a southern group located in the Eastern Mediterranean basin and a northern group located in the Western Mediterranean basin. Gene flow between the two groups is probably restricted, which reveals the complexity of the genetic relationships among populations living in the two basins. This divergence was mainly a consequence of the unequal distribution of some alleles and a cline of frequencies in other ones, which were correlated with substantial differences in genetic diversity among regions. The comparison of the genetic diversity detected within populations (H between 0.0556±0.0246 and 0.1857±0.0648) to the range quoted for this species in previous reports revealed that the southern populations presented a similar values to those recorded in Mediterranean and Atlantic populations, whereas the northern populations showed a higher values.