As the dominant indigenous minority in southern China, Hmong-Mien-speaking Miao people were thought to be the descendants of Neolithic Yangtze rice farmers. However, the fine-scale population structure and genetic profile of the Miao populations remain unclear due to the limited Miao samples from southern China and Southeast Asia. We genotyped 19 individuals from the two largest Miao tribes in Guizhou Province (Southwest China) via SNP chips and co-analyzed the data with published modern and ancient East Asians. The Guizhou Miao displayed a closer genomic affinity with present-day and Neolithic to Iron Age southern East Asians (SEAs) than with most northern East Asians (NEAs). The genetic substructure within Miao groups was driven by different levels of genetic interaction with other ethnolinguistic groups: Hunan Miao (Central China) harbored higher proportions of NEA-related ancestry; Guizhou Miao (Southwest China) and Vietnam Miao (mainland Southeast Asia) received additional gene flow mainly from surrounding groups with Tai-Kadai–related ancestry. There were also more complex admixture events in the newly studied groups between Guizhou Xijiang Miao and surrounding populations compared with Guizhou Congjiang Miao. The qpAdm model further demonstrated that the primary ancestry of Hunan Miao, Guizhou Miao studied here, and Vietnam Miao derived from ancient SEA-related ancestry (represented by coastal early Neolithic SEA Liangdao2), with the additional gene flow from ancient NEA-related ancestry (represented by spatiotemporally inland Yellow River farmers), with slightly different proportions. Our genomic evidence reveals the complex and distinct demographic history of different Miao tribes.