Only 18S rDNA sequences of Sarcocystis spp. in South American camelids (SACs) are deposited in GenBank as references, and the definitive host of S. masoni in SACs is still unclear. Here, S. masoni sarcocysts detected in an alpaca (Vicugna pacos) in China were investigated with the aid of light (LM) and transmission electron (TEM) microscopy, and characterized using four genetic markers, i.e., 18S rDNA, 28S rDNA and ITS, and the mitochondrial cox1. Additionally, the life cycle of the parasite was completed via experimental animal infection. Under LM, S. masoni sarcocysts exhibited numerous 1.3-2.1 μm conical protrusions. Under TEM, the sarcocyst wall contained conical, cylindrical, or irregular-shaped villar protrusions, similar to type 9j. Two dogs (Canis familiaris) fed S. masoni sarcocysts shed sporocysts with a prepatent period of 8-9 days. The newly obtained 18S rDNA sequences showed 98.4-100% identity with those of S. masoni in SACs previously deposited in GenBank. Interestingly, the newly obtained sequences of 18S rDNA and mitochondrial cox1 shared 99.6-100% and 98.2-98.5% identity, respectively, with those of S. cameli in dromedary camels (Camelus dromedaries). Phylogenetic analysis based on sequences of 18S rDNA, 28S rDNA, or mitochondrial cox1 revealed that S. masoni has a close relationship with Sarcocystis spp. in ruminants. The relationship between S. masoni and S. cameli deserves to be further clarified in the future.

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