There is considerable ethno-linguistic and genetic variation among human populations in Asia, although tracing the origins of this diversity is complicated by migration events. Thailand is at the center of Mainland Southeast Asia (MSEA), a region within Asia that has not been extensively studied. Genetic substructure may exist in the Thai population, since waves of migration from southern China throughout its recent history may have contributed to substantial gene flow. Autosomal SNP data were collated for 438,503 markers from 992 Thai individuals. Using the available self-reported regional origin, four Thai subpopulations genetically distinct from each other and from other Asian populations were resolved by Neighbor-Joining analysis using a 41,569 marker subset. Using an independent Principal Components-based unsupervised clustering approach, four major MSEA subpopulations were resolved in which regional bias was apparent. A major ancestry component was common to these MSEA subpopulations and distinguishes them from other Asian subpopulations. On the other hand, these MSEA subpopulations were admixed with other ancestries, in particular one shared with Chinese. Subpopulation clustering using only Thai individuals and the complete marker set resolved four subpopulations, which are distributed differently across Thailand. A Sino-Thai subpopulation was concentrated in the Central region of Thailand, although this constituted a minority in an otherwise diverse region. Among the most highly differentiated markers which distinguish the Thai subpopulations, several map to regions known to affect phenotypic traits such as skin pigmentation and susceptibility to common diseases. The subpopulation patterns elucidated have important implications for evolutionary and medical genetics. The subpopulation structure within Thailand may reflect the contributions of different migrants throughout the history of MSEA. The information will also be important for genetic association studies to account for population-structure confounding effects.