ABSTRACT Southern China is the birthplace of rice-cultivating agriculture, different language families, and human migrations that facilitated these cultural diffusions. The fine-scale demographic history in situ , however, remains unclear. To comprehensively cover the genetic diversity in East and Southeast Asia, we generated genome-wide SNP data from 211 present-day Southern Chinese and co-analyzed them with more than 1,200 ancient and modern genomes. We discover that the previously described ‘Southern East Asian’ or ‘Yangtze River Farmer’ lineage is monophyletic but not homogeneous, comprising four regionally differentiated sub-ancestries. These ancestries are respectively responsible for the transmission of Austronesian, Kra-Dai, Hmong-Mien, and Austroasiatic languages and their original homelands successively distributed from East to West in Southern China. Multiple phylogenetic analyses support that the earliest living branching among East Asian-related populations is First Americans (∼27,700 BP), followed by the pre-LGM differentiation between Northern and Southern East Asians (∼23,400 BP) and the pre-Neolithic split between Coastal and Inland Southern East Asians (∼16,400 BP). In North China, distinct coastal and inland routes of south-to-north gene flow had established by the Holocene, and further migration and admixture formed the genetic profile of Sinitic speakers by ∼4,000 BP. Four subsequent massive migrations finalized the complete genetic structure of present-day Southern Chinese. First, a southward Sinitic migration and the admixture with Kra-Dai speakers formed the ‘Sinitic Cline’. Second, a bi-directional admixture between Hmong-Mien and Kra-Dai speakers gave rise to the ‘Hmong-Mien Cline’ in the interior of South China between ∼2,000 and ∼1,000 BP. Third, a southwestward migration of Kra-Dai speakers in recent ∼2,000 years impacted the genetic profile for the majority of Mainland Southeast Asians. Finally, an admixture between Tibeto-Burman incomers and indigenous Austroasiatic speakers formed the Tibeto-Burman speakers in Southeast Asia by ∼2,000 BP.