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Objective: Studies on population genetics have become highly relevant for understanding the evolutionary history of human settlement in southern South America. The eastern Pampa-Patagonia transition is an area that stands out due to its complex population dynamics, especially during the last about 1,000 years BP. The aim of this work is to characterize the maternal lineages of individuals buried in the Paso Alsina 1 archaeological site (ca. 500 years BP) through the analysis of mitochondrial genetic variability, in order to discuss the population models previously proposed for the southern cone of South America., Methods: Mitochondrial HyperVariable Region I sequences were analyzed on teeth belonging to 20 adult individuals. Statistical analyses were carried out to compare the interpopulation and intrapopulation molecular variability between the results obtained in this work and those previously published data from pre-Hispanic human groups. D1 haplotype network was constructed drawing from data on ancient and extant population group samples., Results: Thirteen sequences (65%) were obtained from the 20 analyzed samples. The maternal lineages or subhaplogroups identified were D1g (69.24%), C1 (15.38%), D1 (7.69%), and D1j (7.69%). There was low haplotype variability within the site; some individuals could be matrilineally related., Discussion: The subhaplogroups registered in Paso Alsina 1 site are in accordance with those reported for ancient and contemporary Patagonian populations. The results suggest that an initial nucleus of individuals carrying mostly subhaplogroup D1g settled in northern Patagonia, from which local diversity of this matrilineage could have arisen. The existence of gene flow in the final late Holocene with groups from Northern Andean Patagonia, as well as from Central Argentina, is proposed. The D1j variant probably developed in the latter region., (© 2020 Wiley Periodicals, Inc.)