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Background: Blastocystis is one of the most common intestinal pathogens in humans and a great number of animals, including sheep and goats. High prevalence and multiple subtypes of Blastocystis have been reported in sheep in several regions of China and abroad. Tibetan sheep is an important economic species for herdsmen in the Qinghai-Tibetan Plateau areas. However, there is a dearth of knowledge about Blastocystis in Tibetan sheep. Here, we detected the prevalence and subtype distribution of Blastocystis in Tibetan sheep.Methods: A total of 761 fecal samples were collected from Tibetan sheep in seven counties of Qinghai Province, northwestern China, and were examined for the prevalence and subtypes of Blastocystis using molecular technology based on the partial small subunit ribosomal RNA gene of Blastocystis.Results: The overall prevalence of Blastocystis in the investigated Tibetan sheep was 10.12% (77/761), and significant differences in prevalence were observed among ruminants from the seven counties (P < 0.01, χ2 = 323.03, df = 6) and across four seasons (P < 0.01, χ2 = 113.91, df = 3). Among them, the highest prevalence was observed among the animals from Haixi County (62.7%) and in summer (28.8%). Sequence analysis revealed three animal-adapted subtypes (ST14, ST10, and ST12) of Blastocystis sp. in these Tibetan sheep, with ST14 as the predominant subtype.Conclusions: To our knowledge, this is the first report on Blastocystis infection in Tibetan sheep, and our findings provide fundamental data for the establishment of effective measures to control this pathogen in Tibetan sheep as well as in other animals.