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Native chickens play at least four significant roles for Thai people. In this study, the genetic variation of native chickens in northeastern Thailand was quantified based on the mitochondrial DNA HVS-I sequences. The phylogenetic relationship between these Thai native chickens as well as those of other geographic regions was also evaluated. A high level of genetic variation was found in the native chickens from northeastern Thailand, somewhat similar to that reported in Indian and Vietnamese populations but higher than African and Chinese native chickens. This likely implies a greater effective population size and longer demographic history and is consistent with the hypothesis that Thailand is within one of the places where chickens were first domesticated. Phylogenetic analyses revealed that Thai native chickens were divided into three lineages. The first lineage was closely related to G. gallus gallus, G. g. domesticus and G. g. murghi whilst the other two lineages were closely related to G. g. spadiceus. Thus, at least three subspecies, G. g. gallus, G. g. murghi and G. g. spadiceus, are likely to have been involved in chicken domestication in Thailand.