To investigate the genetic diversity, clustering relationships and genetic differentiation of Tibetan yak (Bos grunniens) populations, the study analyzed the complete sequence of the mitochondrial DNA ND6 gene of 150 individuals from 15 yak populations to determine the polymorphic loci and the number of haplotypes calculate the haplotype diversity (Hd) and nucleotide diversity (Pi) and to construct the molecular cluster diagram and the haplotype phylogenetic tree. Results showed sequence length of all Tibetan yak mtDNA ND6 was 528 bp and mean percentages of T, C, A and G basic groups were 42.2%,7.6%,20.9% and 29.3%,respectively, revealing certain degree of base composition bias. 37 transitions and 2 transversions were detected in the sequence variation, showing a strong bias of the conversion. According to the variation between the sequences, there were totally 7 type single times in Tibetan yak mtDNA ND6, Hap1 was the mainstream Tibetan yak haplotypes, and the remaining six haplotypes groups as part of the unique. The average haploid type diversity (Hd) and average nucleotide diversity (Pi) were 0.2978 and 0.00191, indicating lower genetic diversity within Tibetan yak populations. The molecular cluster diagram constructed based on genetic distance showed 15 Tibetan yak groups could be divided into two categories. Phylogenetic trees constructed based on the seven kinds of haplotypes indicated that two maternal origin lines existed amons Tibetan yaks.