In this study, the complete sequence of the Tibetan Mastiff mitochondrial genome (mtDNA) was determined, and the phylogenetic relationships between the Tibetan Mastiff and other species of Canidae were analyzed using the coyote (Canis latrans) as an outgroup. The complete nucleotide sequence of the Tibetan Mastiff mtDNA was 16 710 bp, and included 22 tRNA genes, 2S rRNA gene, 13 protein-coding genes and one non-coding region (D-loop region), which is similar to other mammalian mitochondrial genomes. The characteristics of the protein-coding genes, non-coding region, tRNA and rRNA genes among Canidae were analyzed in detail. Neighbor-joining and maximum-parsimony trees of Canids constructed using 12 mitochondrial protein-coding genes showed that as the coyotes and Tibetan wolves clustered together, so too did the gray wolves and domestic dogs, suggesting that the Tibetan Mastiff originated from the gray wolf as did other domestic dogs. Domestic dogs clustered into four clades, implying at least four maternal origins (A to D). The Tibetan Mastiff, which belongs to clade A, appears to be closely related to the Saint Bernard and the Old English Sheepdog.