Genetic variation was surveyed at nine microsatellite loci and the mitochondrial control region (868 bp) to test for the presence of genetic stock structure in young-of-the-year Atlantic bluefin tuna (Thunnus thynnus thynnus) from the Mediterranean Sea. Bluefin tuna were sampled over a period of 5 years from the Balearic and Tyrrhenian seas in the western basin of the Mediterranean Sea, and from the southern Ionian Sea in the eastern basin of the Mediterranean Sea. Analyses of multilocus microsatellite genotypes and mitochondrial control region sequences revealed no significant heterogeneity among collections taken from the same location in different years; however, significant spatial genetic heterogeneity was observed across all samples for both microsatellite markers and mitochondrial control region sequences (FST = 0.0023, P = 0.038 and ΦST = 0.0233, P = 0.000, respectively). Significant genetic differentiation between the Tyrrhenian and Ionian collections was found for both microsatellite and mitochondrial markers (FST = 0.0087, P = 0.015 and ΦST = 0.0367, P = 0.030, respectively). These results suggest the possibility of a genetically discrete population in the eastern basin of the Mediterranean Sea.