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The central Southern Cone of South America was one of the last regions of the globe to become inhabited by people, and remains under-represented in studies of ancient DNA. Here we report genome-wide data from 238 ancient individuals spanning ten millennia. The oldest, from the Pampas region and dating to 10,000 years before present (BP), had distinct genetic affinity to Middle Holocene Southern Cone individuals, showing that differentiation from the central Andes and central east Brazil had begun by this time. Individuals dating to 4,600-150 BP primarily descended from a previously unsampled deep lineage of which the earliest representative is an individual dating to around 8,500 BP. This central Argentina lineage co-existed with two other lineages during the Mid-Holocene and, within central Argentina, this ancestry persisted for thousands of years with little evidence of inter-regional migration. Central Argentina ancestry was involved in three distinct gene flows: it mixed into the Pampas by 3,300 BP and seemingly became the main component there after 800 BP, with central Andes ancestry in northwest Argentina, and with tropical and subtropical forest ancestry in the Gran Chaco. In northwest Argentina, there was an increased rate of close-kin unions by 1,000 BP, paralleling the pattern in the central Andes. In the Paraná River region, a 400 BP individual with a Guaraní archaeological association clusters with Brazilian groups, consistent with Guaraní presence by this time.