The movement of Atlantic bluefin tuna (ABFT) across international boundaries necessitates traceability strategies that would provide more accurate information needed for stock assessment. The Mediterranean Sea is one of the main contributors to ABFT reproduction and global population genetic diversity. In the present study this genetic variability was investigated using 193 samples of adult bluefin tuna from Spain, Turkey and Malta - a longitudinal distance of 3400 km. Analysed were 13 microsatellite loci (eight of which were newly-tested) as genetic markers for the population study. Allele richness measured per locus and sampling location varied from 1.89 to 8.88, taking into account rarefaction. ABFT private alleles were detected in each of the three sampling sites. No significant spatial genetic divergence was found between pairs at the studied locations (F-ST values <0.0001; P-values >0.05). Bayesian clustering analysis corroborated a single and panmictic ABFT population in the Mediterranean Sea. Statistical power analyses indicated a high probability of detecting genetic differentiation and population structure with the sample size and microsatellites used, even at an F-ST value of 0.005. From the results it may be postulated that migrating ABFT during the spawning season are allowing gene flow within the Mediterranean Sea. The complex interplay of movements, including plasticity in the selection of spawning sites with increasing age and environmental conditions, require multiple and new fisheries monitoring and management techniques in order to target the ABFT long-term conservation effectively.