Adenoviruses are common pathogens infecting a wide range of vertebrates. Few cetacean adenoviruses have been described in the literature, and their pathogenicity is still unclear. Using PCR-based viral and bacterial pathogen surveillance in Bering-Chukchi-Beaufort seas bowhead whales (Balaena mysticetus) legally harvested 2012–15 during Alaskan aboriginal subsistence hunts, six of 59 bowhead whales (10%) tested positive for adenovirus DNA in the spleen. We found a high degree of sequence divergence from other mastadenoviruses, suggesting these may represent a novel species, tentatively named bowhead whale adenovirus. The sequences detected are distinct from adenoviruses previously identified in bottlenose dolphins (Tursiops truncatus) and harbor porpoises (Phocoena phocoena), forming two distinct clades in the cetacean hosts. The clinical impact is unclear, since no histopathologic evidence of adenovirus-associated disease was found. Furthermore, detection of adenovirus DNA in the spleen, contrary to other cetacean adenoviruses detected in the intestinal tract, may suggest a broader tissue tropism. Our study demonstrates adenovirus infection in bowhead whales and the usefulness of molecular diagnostics to discover and genetically characterize novel viruses in marine mammals.