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There is considerable confusion concerning the relationships among species of Sarcocystis found in donkeys and horses. Here, we describe a Sarcocystis species in Chinese donkeys (Equus asinus). Sarcocysts were found in 12 of 32 (37.5%) adult donkeys. By light microscopy, they were divided into two types, thin-walled and thick-walled. The thin-walled were macroscopic (up to 320 μm wide) and had short club-like protrusions (up to 2.7 μm long); the thick-walled were microscopic (up to 135 μm wide) and had villar protrusions (up to 5.4 μm long). Ultrastructures of the two types exhibited similar morphological characteristics, including bundled microtubules in the core of the villar protrusions penetrating diagonally into the ground substance, similar to wall type 11c. Three genetic markers, 18S rDNA, 28S rDNA, and mitochondrial cox1, obtained from the two morphotypes were sequenced and analyzed. The sequences of the three loci in the two morphotypes presented high intraspecific similarities of 97.2–99.5%, 97.8–99.6% and 99.0 − 99.9%, respectively. The most similar sequences in GenBank to the newly obtained 18S rDNA, 28S rDNA and cox1 sequences were those of Sarcocystis spp. in horses, with similarities of 90.0 − 97.5%, 94.7 − 95.1%, and 82.6 − 84.5%, respectively. Phylogenetic analysis using the three genetic markers indicated that the Sarcocystis sp. in donkeys formed an individual clade most closely related to a clade encompassing Sarcocystis spp. in horses. Further studies are needed for taxonomic identification of sarcocysts in donkeys because the Sarcocystis species in donkeys and horses are not successfully cross transmissible despite morphological similarities. [ABSTRACT FROM AUTHOR]