ABSTRACT: Donkeys are one important livestock in China because of their nourishment and medical values. To investigate the genetic diversity and phylogenetic relationships of Chinese donkey breeds, a panel of 25 fluorescently labeled microsatellite markers was applied to genotype 504 animals from 12 Chinese donkey breeds. A total of 226 alleles were detected, and the expected heterozygosity ranged from 0.6315 (Guanzhong) to 0.6999 (Jiami). The mean value of the polymorphism information content, observed number of alleles, and expected number of alleles for all the tested Chinese donkeys were 0.6600, 6.890, and 3.700, respectively, suggesting that Chinese indigenous donkeys have relatively abundant genetic diversity. Although there were abundant genetic variations found, the genetic differentiation between the Chinese donkey breeds was relatively low, which displayed only 5.99?% of the total genetic variance among different breeds. The principal coordinates analysis clearly splits 12 donkey breeds into two major groups. The first group included Xiji, Xinjiang, Liangzhou, Kulun, and Guanzhong donkey breeds. In the other group, Gunsha, Dezhou, Biyang, Taihang, Jiami, Qingyang, and Qinghai donkeys were clustered together. This grouping pattern was further supported by structure analysis and neighbor-joining tree analysis. Furthermore, genetic relationships between different donkey breeds identified in this study were corresponded to their geographic distribution and breeding history. Our results provide comprehensive and precise baseline information for further research on preservation and utilization of Chinese domestic donkeys.