In the paper cytochrome oxidase subunit I (COI) in minute gastropods Ecrobia has been sequenced from five localities at the Black Sea (Pomorie Lake and Constanza) and eastern Mediterranean (Gulf of Corinth, Evvoia Island, Attica). Pomorie Lake, a hyperhaline lagoon in eastern Bulgaria, harbours a population of Ecrobia maritima, whose shell and penis morphology are presented. COI partial sequences of E. maritima from Pomorie Lake differ markedly from those in the two Aegean populations not studied so far (Evvoia Island, Attica), and from another four studied earlier, scattered across the Black Sea and Aegean Sea. Such a high level of divergence was unexpected, since Pomorie Lake is only about 20 km away from the closest known locality of E. maritima (Burgas, Bulgaria). The mean p distance within E. maritima was 0.0113. A similar value of p distance (0.0137) was found within the clade grouping all the haplotypes of E. ventrosa. Mean genetic distance between these two species is p = 0.048. E. ventrosa inhabits western Europe, Tunisia, the Peloponnesus and the Corinthian Gulf (Itea: present study), both in the Ionian Sea, and — surprisingly — the coast of the Black Sea in Romania (Constanza: present study). E. maritima was found in the Black Sea and the Aegean Sea. Estimated time of divergence between the species is 3.30 ± 0.23 Mya, and 1.00 ± 0.11 Mya between E. maritima from Pomorie Lake and the other studied localities. The observed pattern is discussed in the context of the geological history of the region, especially glaciation events. Speciation of E. maritima in the Ponto-Caspian waters isolated from the Mediterranean in the Late Piacenzian and divergence of the population presently occurring in Pomorie Lake during the local Calabrian salinity crisis are postulated.