Genetic variation of mussels Mytilus galloprovincialis in central–eastern Mediterranean Sea is investigated in this study. A total of 550 individuals sampled from two cultured and 11 wild populations from Italy, Croatia, Greece and Turkey were genotyped at 10 microsatellite loci. Significant deviations from Hardy–Weinberg expectations were observed in more than 75% of the tests performed. All populations showed extensive heterozygote deficits, which remained at significant levels even after correction for null alleles, providing evidence that null alleles were only partly responsible for deviations from Hardy–Weinberg equilibrium in these molluscs. Moreover, null alleles seem to have limited influence on the population genetic differentiation. Similar levels of multi-locus heterozygosity and allelic richness were observed in all populations, cultured and wild, implying the sustainability of the exploited populations. Lack of isolation by distance and markedly low genetic differentiation between the nine Greek sampling sites (shoreline >1000 km) was revealed by Mantel tests, FST values, exact tests and analyses of molecular variance, indicating that mussels from these regions are either at or close to panmixia. Similarly, patterns of genetic homogeneity were also found between the two Italian samples, whereas the observed genetic differentiation of the populations from Turkey and Croatia probably reflects the specific topographic and oceanographic conditions of these regions.