Genetic differentiation and phylogenetic relationships of big-scale sand smelt Atherina boyeri specimens from 23 sampling sites in Greece and one from a lake in Turkey were investigated. A total of 2180 base pairs (bp) corresponding to the partial sequence of the mitochondrial gene cytochrome oxidase I (coI), cytochrome b (cytb) and control region, as well as of the nuclear protein-coding gene rhodopsin (rh), were determined for the 143 specimens studied. Phylogenetic analysis of each gene segment separately and of the combined dataset revealed the existence of three different and well divergent lineages in Greece. The first corresponds to the lagoon form, in which a clear distinction between the Aegean and Ionian Sea was observed. The other two correspond to the punctuated and non-punctuated marine forms that have been previously reported. The fact that in the present study a population without black spots on the flanks is clustered with the punctuated form and vice versa, however, suggests that differences in colour pattern do not seem to be a sufficient marker to discriminate the two marine forms. In contrast, the presence of a different length insertion between transfer RNA (tRNA) glutamic acid (tRNAglu) and cytb genes in the lagoon and in one of the two forms of marine populations, and its absence from the rest marine-form specimens confirms that this character is conserved and capable to be used for distinguishing the different forms. Even though the divergence values among the different forms were high, their phylogenetic relationships were not able to be resolved.