The long-finned pilot whale, Globicephala melas, is a social, pelagic odontocete distributed widely in the cold temperate waters of the North Atlantic. Despite genetic, morphometric, physiological and observational studies, it remains unclear whether any population substructure exists. We have used eight highly polymorphic microsatellite loci to analyse samples from four disparate sampling sites: USA East Coast (Cape Cod), West Greenland, the Faeroe Islands and the UK. Our results indicate that substructure does exist, and is particularly pronounced between West Greenland and other sites. The magnitudes of the various pairwise comparisons do not support a simple isolation-by-distance model. Instead, the patterns of genetic differentiation suggest that population isolation occurs between areas of the ocean which differ in sea surface temperature. Such a mechanism is supported by the observation that temperature is a primary factor determining the relative distributions of two short-finned pilot whale (G. macrorhynchus) populations off the Pacific coast of Japan.