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The Black Sea is a semi-enclosed body of water that differs from the adjacent Mediterranean Sea in terms of its biodiversity, oceanographical and ecological characteristics. There is growing international concern about pollution in the Black Sea and other anthropogenic threats to its fauna. The bottlenose dolphin (Tursiops truncatus) is one of three species of cetaceans living in the Azov-Black Sea basin. Despite considerable research on bottlenose dolphins elsewhere, the extent of human impacts on the Black Sea populations is unknown. Previous attempts to award special conservation status to Black Sea cetaceans have failed specifically because policy makers have viewed their ecological and evolutionary uniqueness as equivocal. This study assessed divergence between Black Sea, Mediterranean Sea and Atlantic Ocean bottlenose dolphins for 26 cranial measurements (n = 75 adult bottlenose dolphin skulls) and mitochondrial DNA (n = 99 individuals). Black Sea bottlenose dolphins are smaller than those in the Mediterranean, and possess a uniquely shaped skull. As in a previous study, we found the Black Sea population to be genetically distinct, with relatively low levels of mtDNA diversity. Population genetic models suggest that Black Sea bottlenose dolphins have so little gene flow with the Mediterranean due to historical isolation that they should be managed separately.