Hanging Coffin is a unique and ancient burial custom that has been practiced in southern China, Southeast Asia, and near Oceania regions for more than 3,000 years. Here, we conducted mitochondrial whole-genome analyses of 41 human remains sampled from 13 Hanging Coffin sites in southern China and northern Thailand, which were dated between ∼2,500 and 660 years before present. We found that there were genetic connections between the Hanging Coffin people living in different geographic regions. Notably, the matrilineal genetic diversity of the Hanging Coffin people from southern China is much higher than those from northern Thailand, consistent with the hypothesized single origin of the Hanging Coffin custom in southern China about 3,600 years ago, followed by its dispersal in southern China through demic diffusion, whereas the major dispersal pattern in Southeast Asia is cultural assimilation in the past 2,000 years.
The human occupation history of Southeast Asia (SEA) remains heavily debated. Current evidence suggests that SEA was occupied by Hòabìnhian hunter-gatherers until ~4000 years ago, when farming economies developed and expanded, restricting foraging groups to remote habitats. Some argue that agricultural development was indigenous; others favor the "two-layer" hypothesis that posits a southward expansion of farmers giving rise to present-day Southeast Asian genetic diversity. By sequencing 26 ancient human genomes (25 from SEA, 1 Japanese Jōmon), we show that neither interpretation fits the complexity of Southeast Asian history: Both Hòabìnhian hunter-gatherers and East Asian farmers contributed to current Southeast Asian diversity, with further migrations affecting island SEA and Vietnam. Our results help resolve one of the long-standing controversies in Southeast Asian prehistory.