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Bottlenose dolphins are known to have high dispersal capabilities that could lead to genetic connectivity. However, local resident populations are found worldwide as happens in southern Galicia (NW Spain). The aim of this research is to explore whether population substructure and habitat segregation do exist within the Galician bottlenose dolphin community. In the present study, samples from 43 bottlenose dolphins stranded or by-caught in Galicia between 1994 and 2008 were genotyped at 10 microsatellite loci and sequenced at the highly variable mitochondrial control region. In addition, variability of stable isotope ratios (δ13C and δ15N) was assessed for 43 dolphins (5 calves, 38 juveniles and adults) stranded or by-caught in Galicia between 1998 and 2007. Genetic results highlighted the existence of two populations in the area and dolphins from southern Galicia were assigned to a single genetic group. Seven dolphins were classified as possible migrants between putative populations as their genetic makeup did not correspond with their geographical stranding location. Values of δ13C and δ15N were significantly different between the two populations, suggesting resource specialization and partitioning. Dolphins from southern Galicia (inhabiting coastal inlets) showed more variation in their diet, higher trophic levels and greater δ13C compared to animals from northern Galicia (present in more open waters). The existence of fine scale population substructure should be considered in the future designation of Special Areas of Conservation (SACs) for the species as required by the European Habitats Directive.