The existence of nearshore and offshore populations of the bottlenose dolphin has been documented throughout its range. In several cases the two regional forms have been shown to be morphologically distinct, although there is considerable overlap for most characters. The populations off the eastern coast of North America have been the subject of a long-term programme of research on their distribution and movements. In this study, we compare mitochondrial and nuclear genetic markers between dolphins classified as either nearshore or offshore type. These putative populations were found to be distinct at both nuclear and mitochondrial genetic markers. Further, the level of variation among the nearshore dolphins was reduced compared with the offshore population. A broader geographical comparison suggests a shared lineage between offshore dolphins from the western North Atlantic and both offshore and nearshore dolphins from the eastern Atlantic. These results are consistent with local differentiation based on habitat or resource specialization in the western North Atlantic, and suggest differences in the character of the nearshore/offshore distinction in different parts of the world.