Integration of ecological and genetic approaches is a particularly powerful strategy to identify natural population diversity and structure over different timescales. To investigate the potential occurrence of population differentiation in long-finned pilot whales Globicephala melas in the North Atlantic, both biogeochemical (fatty acids and stable isotopes) and genetic (mito - chondrial DNA) markers were analyzed in animals from 4 regions within the North Atlantic: the northwestern Iberian Peninsula, the United Kingdom, the Faroe Islands and the United States of America. Genetic data revealed strong regional levels of divergence, although analysis of molecular variance revealed no differentiation between the northeastern and northwestern Atlantic. Results from biogeochemical tracers supported previous dietary studies, revealing geographic and ontogenetic dietary variation in pilot whales. Fatty acids revealed ecological differentiation between all regions analyzed, while stable isotopes showed an overlap between some sampling regions. These results suggest that both ecological and genetic factors may drive the levels of pilot whale differentiation in the North Atlantic. The ecological differentiation observed may be related to the exploitation of different foraging niches (e.g. oceanic vs. coastal), which can be highly influenced by prey distributions or oceanographic phenomena. Genetic differentiation may result from historical or contemporary processes or even limited dispersal mediated through the social structure displayed by this species and potential foraging specialization. These results highlight some problems when assessing population structure across multiple markers and the ecological vs. evolutionary timescales over which differences may accumulate. Notwithstanding, the data provide preliminary information about pilot whale diversity and stocks in the North Atlantic, giving essential baseline information for conservation plans.