In this study, the complete mitochondrial genome sequence of the Tibetan Mastiff was reported. The total length of the mitogenome is 16,729 bp. It contains the typical structure, including 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and 1 control region is in line with other canine animals. We further identified genome-wide variations among different canine mitochondrial genomes and indicated that the D-loop region harbors the most sequence variation, which will provide sequence variation information for the protection and utilization of the Tibetan Mastiff germplasm resource.