Combining over a hundred libraries from a single sample, we provide insight into the genetics of one of the early civilizations of the ancient world.

We report the first ancient DNA data from the Indus Valley Civilization (IVC). The individual we sequenced fits as a mixture of people related to ancient Iranians (the largest component) and Southeast Asian hunter-gatherers, a unique profile that matches ancient DNA from 11 genetic outliers from sites in Iran and Turkmenistan in cultural communication with the IVC. These individuals had little if any Steppe pastoralist-derived ancestry, showing it was not ubiquitous in northwest South Asia during the IVC as it is today. The Iranian-related ancestry in the IVC derives from a lineage leading to early Iranian farmers, herders and hunter-gatherers before their ancestors separated, contradicting the hypothesis that the shared ancestry between early Iranians and South Asians reflects a large-scale spread of western Iranian farmers east. Instead, sampled ancient genomes from the Iranian plateau and IVC descend from different groups of hunter-gatherers between whom farming spread via movement of ideas.