Indigenous peoples of America represent the last principal expansion of humans across the globe1, yet their genetic history remains one of the least explored2. Although these populations have inhabited the continent for thousands of years3, their evolutionary history remains largely unresolved4,5, owing to the limited availability of genomic data. Here we present data on 128 high-coverage Indigenous American genomes and show they harbour extensive and previously uncharacterized genetic diversity, reflecting at least three dispersals into South America, followed by regional differentiation and long-term continuity. We identified widespread natural selection signals in genes associated with immunity, metabolism, reproduction and development, which were shaped by adaptation to diverse environmental conditions. Notably, several genomic regions exhibit a remarkable allele sharing with Australasian populations, probably originating from an ancient admixture event and partly maintained by selection for more than 10,000 years. We also detected distinct contributions from archaic humans with adaptive introgression affecting key biological functions. The limited overlap between the regions of Australasian affinity and archaic ancestry indicates independent evolutionary origins of these signals. These findings challenge simplified models of continental settlements and show a more dynamic and complex evolutionary history for the Indigenous peoples in America.
