Yang, S.-l., Li, A.-P., Xu, L.-x. and Yang, H. 2013. Short Communication: Mitochondrial DNA D-loop sequence diversity and origin of Chinese pony breeds (Equus caballus). Can. J. Anim. Sci. 93: 313-319. Previous studies based on mitochondrial DNA (mtDNA) data have shown that Chinese horses have high genetic diversity. However, little is known about the genetic diversity of mtDNA D-loop sequences and maternal origin of five Chinese pony breeds. In the present study, genetic diversity of 343-bp D-loop sequences for136 individuals representing five Chinese pony breeds was analyzed. To address the question of the single and multiple maternal origin of Chinese pony populations, 13 partial mtDNA D-loop reference sequences from GenBank representing eight ancient and modern horse breeds (Connemara, Garrano, Sorraia, Pottok, Losino, Cheju, Tuva and a Swedish horse) were added to conduct the phylogenetic analyses. A total of 59 haplotypes and 50 polymorphic loci were detected, the haplotype diversity (h) ranged from 0.894 to 0.947 and nucleotide diversity (p) ranged from 0.0184 to 0.0229, suggesting relatively abundant genetic diversity in the Debao, Yunnan and the Guizhou breeds. The phylogenetic tree and median-joining network show multiple maternal origins of the five Chinese pony breeds.