Abstract: Tibetan Horse lives in Qinghai-Tibet Plateau of South-West China, the controversy on whether Tibetan Horse should be classified into Southwest Horse Type or not has existed in China for a long time as there are four other type horses in the country. In the study, the genetic diversity and structure of Tibetan Horse were investigated using 14 microsatellite markers and 153 animals belonging to three Tibetan Horse subpopulations in Xigaze, Qamdo and Nagqu, respectively and Mongolian Horse and Ningqiang Pony which served as reference groups to Tibetan Horse. The results showed that the mean Number of Allele (MNA) across the 14 loci in Tibetan Horse was 13.1 and the average observed heterozygosity (0.710) and PIC (0.752) were slightly higher than those (0.692 and 0.773, respectively) of Mongolian Horse. Although, there was abundant genetic variation, genetic differentiation between horse populations was low, with only 2.1% of the total genetic variance among populations. Qamdo, Xigaze and Mongolian Horse had close relationship and Nagqu is distinctly separated from the other two Tibetan Horse subpopulations and referenced breeds in the clusters. The results indicated the genetic relationship of Tibetan Horse were closer to Mongolian Horse rather than to Ningqiang Pony, a typical breed in Southwest Horse Type, which suggests that Tibetan Horse was a unique horse breed which could not be classified into Southwest Horse Type.