Tai-Kadai (TK) is one of the major language families in Mainland Southeast Asia (MSEA), with a concentration in the area of Thailand and Laos. Our previous study of 1234 mtDNA genome sequences supported a demic diffusion scenario in the spread of TK languages from southern China to Laos as well as northern and northeastern Thailand. Here we add an additional 560 mtDNA genomes from 22 groups, with a focus on the TK-speaking central Thai people and the Sino-Tibetan speaking Karen. We find extensive diversity, including 62 haplogroups not reported previously from this region. Demic diffusion is still a preferable scenario for central Thais, emphasizing the expansion of TK people through MSEA, although there is also some support for gene flow between central Thai and native Austroasiatic speaking Mon and Khmer. We also tested competing models concerning the genetic relationships of groups from the major MSEA languages, and found support for an ancestral relationship of TK and Austronesian-speaking groups.