The mtDNA hypervariable segment I sequences (HVS I) were sequenced in 64 Shuis from Guizhou Province. 73 sites were polymorphic in the 495 bp fragment that sequenced, identified 48 different haplotypes. Phylogenetic analysis of the haplotypes suggested that there were some ancestral haplotypes in current Shuis, and these haplotypes were also present in Eurasia populations as well as in other ethnic groups. Demographic analysis of the Shuis demonstrated a unimodal distribution that is typical for a population undergone expansion in the past and with a high Tau value, which suggested that the Shui group might be a ancestral population. Combined with the analysis of the reported data, the Shui ethnic group showed a generally similar genetic component with the Zhuang from Guangxi, but it is also different from these typical south populations as Zhuang, Cantonese.