Bottlenose dolphins (Tursiops truncatus) have a world-wide distribution, and show morphotypic variation among regions. Distinctions between coastal and pelagic populations have been documented; however, regional patterns of differentiation had not been previously investigated in a wider geographic context. We analysed up to nine different populations from seven different areas of the world by mitochondrial DNA and microsatellite DNA markers, and found differentiation among all putative regional populations. Both mtDNA and microsatellite DNA data show significant differentiation, suggesting restricted gene flow for both males and females. Dolphins in coastal habitat showed less variability and were in most cases differentiated from a pelagic lineage, which could suggest local founder events in some cases. Two coastal populations recently classified as belonging to a new species, T. aduncus, were each highly differentiated from populations of the truncatus morphotype, and from each other, suggesting a possible third species represented by the South African aduncus type.