Geographic variations of Truchurus trachurus based on mitochondrial 16S rDNA gene from 8 locations, including the Black, Marmara, Aegean and north-eastern Mediterranean Seas, were investigated. Restriction fragment length polymorphism (RFLP) analyses revealed 14 different composite haplotypes for 307 individuals and diagnostic restriction sites for discriminating among populations. The distribution of haplotypic groups mostly followed the geographic origin of populations. Average haplotype diversity within populations was high (0.7311), and nucleotide diversity was low (0.0071). Mean nucleotide divergence among samples of T. trachurus was 0.00271. The highest value of pairwise inter-group nucleotide divergence was detected between the West and East Black Sea samples (0.01119), and the lowest (-0.00018) between the two North-eastern Mediterranean samples. In Monte Carlo pairwise comparisons of haplotype frequencies genetically different populations were detected. The distribution of haplotypes and the pairwise estimate of nucleotide divergence exposed high differentiation of the Black Sea (BS2) population with respect to the others. Mantel's test showed that the genetic distances between these Populations were not associated with their geographical distances (r = 0.326; P > 0.05).