To investigate the genetic diversity and maternal origin of Huaibei Grey donkey, 10 Huaibei Grey donkeys were randomly selected from the large group, and their mtDNA D-loop sequences of were analyzed by PCR amplification and sequencing technique, and compared with those of some existing donkey breeds sequences in GenBank and explored the genetic diversity and maternal origin of Huaibei Grey donkey. The results showed that 407 bp D-loop base sequences were obtained, 28 polymorphic sites, including 8 conversions and 1 transversion were detected, and the Pi, Hd and K were 0.021 95, 0.778 and 8.911, respectively, indicating abundant maternal genetic diversity of the Huabei Grey donkey. From the analysis of five haplotypes of nucleotide sequences in the D-loop region, it was found that the Huabei Grey donkey might have two maternal origins, and genetic distance indicated that the genetic distance between the Huabei Grey donkey and the Croatian domestic donkey was closer.The results of the present study provide a reference for genetic resources conservation and exploitation of the genetic resources of the Huabei Gray donkey at the molecular level.