Beaked whales, including northern bottlenose whales ( Hyperoodon ampullatus ), are widely considered deep-diving squid specialists. In Canada, where northern bottlenose whales are of conservation concern following historical depletion, resolving diet composition is critical for understanding differences in population recovery and evaluating risks from overlap with regional fisheries. Here, we analyse stable isotopes (δ¹³C, δ¹⁵N; Bayesian mixing models) from skin ( n = 141 samples) and fatty acids from blubber (quantitative fatty acid signature analysis, QFASA; n = 91 samples) from northern bottlenose whales, alongside a large library of potential prey (19 species) collected across the western North Atlantic (42–74 °N). Stable isotope data suggested that females in both Arctic and Scotian Shelf populations exhibited broader ecological niches than males. However, diet composition differed between regions, mirroring known genetic structure. In Arctic whales, squids’ contribution was low, while diets were predominantly fish-based, with contributions from grenadiers (family Macrouridae ), redfish ( Sebastes spp.), and Greenland halibut ( Reinhardtius hippoglossoides ). Scotian Shelf whales consumed both demersal fish (redfish) and squid ( Gonatus fabricii ). Both biomarkers indicate substantial reliance on demersal fish, consistent with flexible foraging strategies and suggesting a broader diet than the obligate squid specialist view. These findings underscore the need to consider diverse prey fields and potential fisheries interactions in conservation and recovery efforts. • Biomarkers show regional and seasonal variation in northern bottlenose whale diet • Demersal fishes, including grenadiers, redfish, and Greenland halibut, are key prey • Results indicate a broader diet composition than an obligate squid specialist model • Dietary flexibility highlights the potential for overlap with regional fisheries
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