The Tai-Kadai (TK) language family is thought to have originated in southern China and spread to Thailand and Laos, but it is not clear if TK languages spread by demic diffusion (i.e., a migration of people from southern China) or by cultural diffusion, with native Austroasiatic (AA) speakers switching to TK languages. To address this and other questions, we obtained 1,234 complete mtDNA genome sequences from 51 TK and AA groups from Thailand and Laos. We find high genetic heterogeneity, with

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... 2 haplogroups. TK groups are more genetically homogeneous than AA groups, with the latter exhibiting more ancient/basal mtDNA lineages, and showing more drift effects. Modeling of demic diffusion, cultural diffusion, and admixture scenarios consistently supports the spread of TK languages by demic diffusion. Surprisingly, there is significant genetic differentiation within ethnolinguistic groups, calling into question the common assumption that there is genetic homogeneity within ethnolinguistic groups.