China had the largest population of raising donkeys in the world, however the number of Chinese indigenous donkey decreased dramatically due to the increase of agriculture mechanization in the last century. The species has still been important in China because of its edible and medical value, therefore the survey on its genetic diversity in China is necessary for its conservation and utilization. In this study, 15 microsatellite markers were used to evaluate genetic diversity and population structure of five Chinese indigenous donkey breeds. The mean values of expected heterozygosity, allelic richness, and total number of alleles for all the tested Chinese donkeys were 0.70, 6.04, and 6.28 respectively, suggesting that the genetic diversity of Chinese indigenous donkeys is rich. The Bayesian analysis and principal component analysis plot yielded the same clustering result, which revealed that Guanzhong donkey was the most differentiated breed in all detected samples, and Jinnan (JN) and Guangling (GL) were genetically closed together. Additionally, our results indicated that the heterozygote deficit was severe in two Chinese indigenous donkey breeds (GL and JN), and it warned us that animal conservation activities on this species should be considered carefully in near future.