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Abstract ObjectivesThis study aims to characterize the genetic histories of ancient hunter‐gatherer groups in Fuego‐Patagonia (Chile) with distinct Marine, Terrestrial, and Mixed Economy subsistence strategies. Mitochondrial (mtDNA) and Y‐chromosome data were generated to test three hypotheses. H₀: All individuals were drawn from the same panmictic population; H₁: Terrestrial groups first populated the region and gave rise to highly specialized Marine groups by ~7,500 cal BP; or H₂: Marine and Terrestrial groups represent distinct ancestral lineages who migrated independently into the region. MethodsAncient DNA was extracted from the teeth of 50 Fuegian‐Patagonian individuals dating from 6,895 cal BP to after European arrival, and analyzed alongside other individuals from previous studies. Individuals were assigned to Marine, Terrestrial, and Mixed Economy groups based on archeological context and stable isotope diet inferences, and mtDNA (HVR1/2) and Y‐chromosome variation was analyzed. ResultsEndogenous aDNA was obtained from 49/50 (98%) individuals. Haplotype diversities, F_STcomparisons, and exact tests of population differentiation showed that Marine groups were significantly different from Terrestrial groups based on mtDNA (p < 0.05). No statistically significant differences were found between Terrestrial and Mixed Economy groups. Demographic simulations support models in which Marine groups diverged from the others by ~14,000 cal BP. Y‐chromosome results showed similar patterns but were not statistically significant due to small sample sizes and allelic dropout. DiscussionThese results support the hypothesis that Marine and Terrestrial economic groups represent distinct ancestral lineages who diverged during the time populations were expanding in the Americas, and may represent independent migrations into Fuego‐Patagonia.