To investigate the genetic diversity, clustering relationships and genetic differentiation of Tibetan yak (Bos grunniens) populations, we analyzed the complete sequence of the mitochondrial DNA control region (D-loop) of 114 individuals from 11 yak populations. To clarify the origin, phylogeny and taxonomic position of B. grunniens, we retrieved a further 48 D-loop sequences from five diverse populations of B.runniens, resulting in 162 sequences from 16 populations in total. Results showed some nucleotide bias in the 890-896bp bp Tibetan yak mtDNA D-loop sequence, with nucleotide frequencies of 28.5%, 25.3%, 32.4% and 13.8% for T, C, A and G respectively. A total of 90 haplotypes were identified, with 130 polymorphic sites (14.33% of total analyzed sites). Of these, 45 sites represented single nucleotide polymorphisms; while a further 85 sites were parsimony-informative. The population haplotype diversity ranged from 0.93-1.00 with an average of 0.98±0.008, indicating a high level of genetic diversity within Tibetan yak populations. The majority of polymorphisms were A/G and T/C transitions or A/T transversions. Polymorphisms were more frequent in the Sangri yak and Baqing yak populations, and occurred less frequently in the Kangbu yak and Jiali yak populations. The 90 haplotypes were divided into two clusters by constructing a network relationship chart. This result suggests that Tibetan yaks may be derived from two separate maternal lineages. The degree of nucleotide difference among the 11 populations ranged from 0.503%-1.416%. The Sangri and Baqing populations had the highest degree of difference, whereas the lowest difference was observed between the Kangbu and Jiali populations. Because of this data, we suggest an apparent genetic differentiation between the Sangri and Baqing populations. Tajima's test of selective neutrality was significant (0.01 < P<0.05) for the Leiwuqi yak, Dingqing yak, Jiangda yak, and Gongbujiangda yak populations, but not significant (P > 0.05) for other populations. These results suggested that the Leiwuqi,Dingqi,Jiangda,and Gongbujiangda populations may have undergone expansions. The molecular variance among groups of the 16 Bovinae populations, as well as differences observed in the significant test, indicated that Bison bison is likely the ancestor of B. grunniens, and that there may be 2 maternal lineages. There also appears to be a high level of genetic diversity within Tibetan yak populations. The clustering relationship and analysis of molecular variance also suggested that Tibetan yak could be divided into two species. These study results not only provide an important theoretical basis for assessment and selection of genetic diversity in Tibetan yak populations, but are of great significance for elucidating the origin and classification of B. grunniens, and for the conservation of genetic resources.