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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 remains unclear due to the limited Miao samples from Southern China and Southeast Asia. Here, we genotyped 19 individuals from the two largest Miao tribes in Guizhou province (Southwest China) via SNP chips and co-analyzed with published available modern and ancient East Asians. We observed that studied Guizhou Miao displayed a closer genomic affinity with present-day and Neolithic-to-Iron Age Southern East Asians than with most Northern East Asians. 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 Northern East Asians-related ancestry; Guizhou Miao (Southwest China) and Vietnam Miao (mainland Southeast Asia) received the additional gene flow mainly from surrounding Tai-Kadai speaking-related ancestry. Besides, there were more complex admixture events between newly studied Guizhou Xijiang Miao and surrounding populations compared with studied Guizhou Congjiang Miao. The qpAdm model further demonstrated that the primary ancestry of Hunan Miao, studied Guizhou Miao and Vietnam Miao derived from ancient Southern East Asian (SEA)-related ancestry (represented by coastal Early Neolithic SEA Liangdao2) with the additional gene flow from ancient northern East Asian-related ancestry (represented by spatiotemporally inland Yellow River farmers), with slightly different proportions. Conclusively, our genomic evidence revealed the complex and distinct demographic history of different Miao tribes.