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Upper Mesopotamia played a key role in the Neolithic Transition in Southwest Asia through marked innovations in symbolism, technology, and diet. We present 13 ancient genomes (c. 8500 to 7500 cal BCE) from Pre-Pottery Neolithic Çayönü in the Tigris basin together with bioarchaeological and material culture data. Our findings reveal that Çayönü was a genetically diverse population, carrying mixed ancestry from western and eastern Fertile Crescent, and that the community received immigrants. Our results further suggest that the community was organized along biological family lines. We document bodily interventions such as head shaping and cauterization among the individuals examined, reflecting Çayönü’s cultural ingenuity. Last, we identify Upper Mesopotamia as the likely source of eastern gene flow into Neolithic Anatolia, in line with material culture evidence. We hypothesize that Upper Mesopotamia’s cultural dynamism during the Neolithic Transition was the product not only of its fertile lands but also of its interregional demographic connections.

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