Nucleotide sequences of the D-loop region of human mitochondrial DNA from four Yunnan nationalities, Dai, Wa, Lahu, and Tibetan, were analyzed. Based on a comparison of 563-bp sequences in 99 people, 66 different sequence types were observed. Of these, 64 were unique to their respective populations, whereas only 2 types were shared between the Lahu and Wa nationalities. The D-loop sequence variation and phylogenetic analysis suggested that the 99 mtDNA lineages were classified into eight clusters in the phylogenetic tree. All lineages that had a 9-bp deletion in the COII/tRNALYs intergenic region appeared in one cluster in the D-loop tree, suggesting a single event of the deletion in the Yunnan nationalities studied. Genetic distances, based on net nucleotide diversities between populations including Han Chinese and mainland Japanese, revealed that the Dai, Wa, Lahu, and Han Chinese are closely related to each other, while Tibetan and mainland Japanese formed a single cluster. The bootstrap probability of separation between the Dai-Wa-Lahu-Chinese clade and the Tibetan-Japanese clade was 99%, indicating that there are at least two different origins among minority groups in Yunnan province. Although the genetic distance between Tibetan and Japanese within the clade is rather long, the results may shed light on the origins of mainland Japanese.