Genetic diversity of Mithun population in Bhutan was studied using 14 microsatellite markers. Two sets of two-step polymerase chain reactions were performed with multiplex and individual markers for genotyping 105 hair samples collected from Arong in Samdrupjongkhar (AS, 36) and Wangdigang in Zhemgang (WZ, 69). Fifty-three alleles were detected with average of 3.89 alleles and polymorphism information content of 0.44 ± 0.03 per locus. A low level of genetic variability within population was present with observed heterozygosity at 0.50 ± 0.06 and expected heterozygosity at 0.48 ± 0.06. Analysis of molecular variance attributed 58 percent of total variation to within the individuals. Mean F IS and F IT were −0.056 and 0.005 respectively, indicated low level of population differentiation and limited out-breeding. The normal L-shaped distribution of allelic frequencies without any mode-shift revealed the absence of recent genetic bottleneck in Mithun populations. Therefore to manage inbreeding in the small Mithun population of Bhutan, periodic assessment of inbreeding levels and exchange of animals between farms is recommended to reduce frequency of introduction of animals from India.