Mussels (Mytilus spp.) are one of the most cultivated and commercialized bivalves in southern Chile; culture is currently supplied almost entirely from wild-caught seed obtained from relatively few collection centers. The genetic diversity and differentiation of the blue mussel in southern Chile was investigated by sampling six locations: one natural bank and five seed collection centers. Nine polymorphic microsatellite (SSR) loci were genotyped (Mgu1, Mgu3, MT203, MT282, Mg15, Mg56, Med737, MIT02 and MGE005). We found 75 different alleles, six of which were private alleles. Of the analyzed loci, 45 of 54 tests performed deviated from Hardy-Weinberg equilibrium after sequential Bonferroni correction (P < 0.05), revealing significant heterozygote deficiencies. The polymorphic information content (PIC) ranged from 0.322 (MGE005) to 0.893 (Mgu1). Despite the long distance between some sampling sites (up to 1360 km), genetic differentiation among the sites was low (FST = 0.043, P < 0.0001). The Bayesian cluster analysis (STRUCTURE) indicated two probable clusters, while the non-parametric cluster analysis (AWclust) identified two to four clusters. Both analyses showed a high level of admixture within clusters. Our results indicate that blue mussels in southern Chile show lower genetic diversity than in other countries, low inbreeding levels, and limited genetic differentiation among locations.