Wolves were once widely distributed throughout China. The number of Chinese wolves has drastically declined in recent years as a result of environmental destruction and habitat loss. At present, wolves in China are mainly distributed in sparsely populated western and northern regions of the country. To protect this species, an understanding of genetic diversity and phylogenetic relationships of wolf populations in China is necessary. In this study, we analyzed the partial mitochondrial DNA (mtDNA) D-loop region and the Cyt b (Cytochrome b)gene of wolves originating from four distinct geographical populations in Xinjiang, Qinghai, Inner Mongolia, and Jilin. Using standard molecular biology techniques, we generated sequences of the mtDNA control region hypervariable region I (HVR I) from 44 wolves, and sequences of the Cyt b gene from 40 wolves. The resulting 582-bp HVR I sequence alignment contained 51 variable sites, which corresponded to 8.76% polymorphic loci. Similarly, 31 variable sites, all base substitutions, were identified in the aligned Cyt b sequence data set. We detected 16 haplotypes among the 44 HVR I gene sequences. The Inner Mongolian population and the population from Jilin were found to share the same HVR I haplotype. When this data set was combined with previously generated Chinese wolf sequences available in Genbank, a total of 28 haplotypes from approximately 91 Chinese wolves were identified. To explore the genetic diversity of these geographic populations, we calculated nucleotide diversity and haplotype diversity from the HVR I sequences. The population from XinJiang was found to have the highest genetic diversity. When we compared these results with previously reported nucleotide diversities of wolves from other countries and regions, we found that Chinese wolves had the highest nucleotide diversity. In the phylogenetic tree generated using the HVR I haplotypes, Chinese wolves are primarily divided into two lineages. The first lineage comprises haplotypes of wolf populations from the Qinghai-Tibet Plateau and surrounding areas. There is no obvious relationship with geographical structure among the remaining wolf haplotype groups. Considering their high level of genetic diversity and restricted geographic distribution, we speculate that the Qinghai-Tibet Plateau populations have evolved in isolation over a long period of time. To investigate the phylogenetic position of Chinese wolves, we downloaded 123 mtDNA partial control region sequences from Genbank, which were obtained from wolves primarily distributed in Eurasia and North America. In the phylogenetic tree generated from these sequences, wolf populations from the Qinghai-Tibet Plateau are clearly separated from wolves from other regions of China. The results of this phylogenetic analysis suggest that wolf populations in the Qinghai-Tibet Plateau of China comprise an ancient lineage that is still extant in these higher elevation regions. Based on genetic distances calculated using Cty b sequence data, we estimate that the two lineages diverged about 1.1 million years ago, corresponding to the time period during which the Qinghai-Tibet Plateau was suddenly uplifted by geological processes. Based on these results, we propose that the wolves of the Qinghai-Tibet Plateau and the widespread wolf Canis lupus chanco are not the same subspecies.