Cetaceans were an important food and raw material resource for the South American hunter–gatherer–fisher (HGF) communities of Tierra del Fuego. Historic ethnographic evidence suggests that relatively mobile HGF groups came together in large numbers to exploit carcasses from individual cetacean stranding events. Substantial accumulations of whale bones within shell middens in the Lanashuaia locality of the Beagle Channel suggests that these social aggregation events may also have occurred in pre-historic periods. The difficulty in assigning taxonomic identifications to the fragmentary whale remains, however, made it difficult to explicitly test this hypothesis. Here, we applied two different biomolecular techniques, collagen peptide mass fingerprinting (ZooMS) and ancient mitochondrial DNA analysis to 42 archeological bone fragments from the Lanashuaia locality to provide accurate species identifications. There was a clear correspondence between ZooMS and DNA results, identifying five different cetacean species (Southern bottlenose, blue, humpback, right, and sei whale) as well as human and sea lion remains. The biomolecular results were not conclusively consistent with HGF social aggregation, revealing an unexpectedly diverse range of cetaceans within the Lanashuaia middens. However, the results could not fully refute the hypothesis that cetacean remains can be used as anthropic markers of aggregation events, as the observed species and haplotypes revealed potential shared exploitation of some whale resources between midden sites.