Dezhou donkey is one of the excellent large donkey breeds in China. In our study, eight microsatellite markers were used to genotype from each of three populations of Chinese Dezhou donkeys: 67 individuals in Liaocheng (pop1), 103 individuals in Binzhou 1 (pop2), and 102 individuals in Binzhou 2 (pop3), in order to study the genetic diversity of these populations. A total of 213 alleles were detected, and thePICresults showed that eight loci were all highly polymorphic. The means ofHoandHeof pop2 were the highest, which were 0.646 and 0.717, respectively. The PCoA analysis demonstrated that the samples from three conservation farms were mixed together. The phylogenetic tree showed that pop 2 and pop 3 were closely related. The phylogenetic tree results clustered that 272 donkeys were divided into six groups. AMOVA analysis showed that the genetic variation was mainly concentrated within the population and the genetic differentiation among the populations was low. Fst values between populations also indicated that genetic differentiation between populations was too small to be considered. This indicated a low probability of inbreeding within the population. And this also showed that the conservation and breeding of Dezhou donkeys in recent years have achieved excellent results. The investigation of genetic diversity in three Dezhou donkey original breeding farms can provide reference data for the selection and breeding of excellent breeds of Dezhou donkey.