The genetic polymorphism of mitochondrial DNA in the coding region in 16 ethnic populations of Yunnan was analyzed using PCR-RFLP in a total of 654 samples. Seventeen haplogroups were found, four of which were undefined haplogroups. Haplogroup distribution and Principal Component (PC) analysis showed that the ethnic groups descended from Bai-Yue tribe have B, F and M7 as the predominant haplogroups,which indicated their origination from southern China. Haplogroup A, D and N9 were predominant in the Mongolian ethnic group, reflecting their north-originated characteristics. The groups descended from Di-Qiang tribe shared the predominant haplogroups with both the south and north originated groups,which demonstrated that they inherit the maternal characteristics from both southern and northern populations. There is genetic difference among the populations in the same ethnic group and is usually smaller than that among the ethnic groups from different ancient tribes, but not necessarily smaller than that among the groups from the same tribe.