Tibetan Mastiff is one of the most archaic, ferocious and the largest dogs in the world. A total of 140 individuals from four geographically separated populations in China (Tibet, Gansu, Qinghai and Beijing) were sampled and genetic diversity was assessed using 10 microsatellite loci on eight different chromosomes. The mean number of alleles per locus ranged from 6 to 13. The mean observed and expected heterozygosities, polymorphism information content and allelic richness were 0.69, 0.79, 0.76 and 7.59, respectively, indicating relatively high genetic diversity in Tibetan Mastiff. However, a highly significant deficiency in heterozygote was observed within populations (mean FIS = 0.11, bootstrap 95% confidence interval (0.06, 0.17)) and total inbreeding (mean FIT = 0.12, bootstrap 95% confidence interval (0.06, 0.18)), along with strong inbreeding coefficients within populations (Fis > 0.09), all of which suggested that intense inbreeding practices occurred in Tibetan Mastiff. Therefore, effective and appropriate breeding management projects in present Tibetan Mastiff will be desirable and urgent. Low genetic differentiation was obtained with a mean FST of 0.01 (bootstrap 95% confidence interval (0.007, 0.019)). Additionally, the four Tibetan Mastiff populations showed close relationships in the neighbor-joining polygenetic tree based on the coancestral genetic distances. Tibetan Mastiff was investigated by using microsatellite loci at the first time, which could facilitate the better understanding of present situation at the molecular level, breed conservation and utilization in Tibetan Mastiff.