The mitochondrial DNA (mtDNA) D-loop sequences with 399 bp in 26 individuals from 5 donkey breeds in China were analyzed. Aligned by Clustal W software,the results showed that 23 polymorphic nucleotide sites and only transition with the percentage of 5.76% in 399 bp were observed. In reference to mtDNA D-loop sequences of European domestic donkey as a control, the average percentage of mtDNA D-loop nucleotide variation in 5 Chinese donkey breeds was 1.80%. The average percentages of D-loop nucleotide variation from Liangzhou donkey (LZ), Yunnan donkey (YN), Guanzhong donkey (GZ), Xinjiang donkey (XJ) and Jiami donkey (JM) were 0. 35%, 1.25%, 2.30%, 2.91% and 2.20% respectively. The average sequence divergence estimated from D-loop sequences varied from 0.25% - 5.01% within breeds and 4.51% - 5.51% among breeds, respectively, demonstrating that there existed rather abundant mitochondrial genetic diversity in Chinese donkeys. Comparisons of the 26 sequences revealed 11 mitochondrial haplotypes; the percentage of haplotype was 42.31%. This phenomenon demonstrated that the mitochondrial genetic diversity in Chinese donkey breeds is being reduced. It is urgent to protect the genetic resources of Chinese donkey. The molecular phylogenetic tree of mtDNA D-loop sequences in 5 Chinese donkey breeds,6 sequences of Asian wild ass (Equus asinus kiang, Equus asinus kulan, Equus asinus hemionus;) and 4 sequences of European domestic donkeys from GenBank was constructed by Neighbor-Joining method. It was the first time proved in molecular level that the origin of Chinese donkey breeds was from African wild ass (Equus africanus africanus and Equus africanus somaliensis), not from Asian wild ass as bescribed in the paper.