Objective To investigate the prevalence of piroplasm infections and genetic evolutionary characteristics of piroplasm in Tibetan sheep from some areas in Qinghai Province. Methods Whole blood samples were collected from Tibetan sheep across 12 counties (districts) in 8 states (cities) of Qinghai Province, and genomic DNA was extracted using the silicon matrix column method. The 18S rRNA V4 hypervariable region sequences of piroplasm was amplified using nested PCR assay, and samples positive for nested PCR assay were sequenced, followed by sequence alignment using the BLAST program in NCBI to identify the piroplasm species. The sequences of samples positive for nested PCR assay were subjected to haplotype analysis and nucleotide polymorphism analysis using the software DnaSP version 6, and a haplotype network diagram was constructed using the PopArt software. A phylogenetic tree based on the 18S rRNA V4 hypervariable region sequence of piroplasm was built using the maximum likelihood method with the sequence of Babesia microti (GenBank accession number: AB242176) as the outgroup. Results A total of 475 Tibetan sheep blood samples were collected, and 193 blood samples were positive for piroplasm, with 40.6% (193/475) overall prevalence of infections. piroplasm infection was detected across 12 districts (counties), and the three highest prevalence seen in Menyuan (94.1%, 96/102), Haiyan (72.7%, 24/33), and Ledu (47.8%, 11/23). The species of piroplasm infecting Tibetan sheep included Theileria luwenshuni, T. ovis, T. uilenbergi and Theileria sp. OT3, with infection prevalence rates of 20.6% (98/475), 17.7% (84/475), 1.3% (6/475), and 1.1% (5/475), respectively. There were 13 haplotypes of T. luwenshuni, with TlH1 as the dominant haplotype (76.5%, 75/98), which were mainly distributed in Menyuan, Ledu, and Minhe, and there were 9 haplotypes of T. ovis, with ToH1 as the dominant haplotype (85.7%, 72/84), which were prevalent in Haiyan, Zeku, Menyuan, Gangcha, Huangyuan, Dari, Guinan, Tianjun, and Qilian, while there were 4 haplotypes of T. uilenbergi, with TuH2 as the dominant haplotype (3/6), which were mainly distributed in Menyuan, and there were 4 haplotypes of Theileria sp. OT3, with TsH3 as the dominant haplotype (2/5), which were mainly distributed in Minhe. Phylogenetic analysis revealed that the sequence of the T. luwenshuni 18S rRNA V4 hypervariable region from the whole blood of Tibetan sheep in Qinghai Province was clustered into a clade with the sequences of Budorcas taxicolor-derived T. luwenshuni isolate and sheep-derived T. luwenshuni isolates from Lanzhou, Tianzhu, Qinghai and Shaanxi in GenBank, and the sequence of Theileria sp. OT3 was clustered into a clade with those of Theileria sp. OT3 from yak-derived parasite isolates in Qinghai, goat-derived strains in Shaanxi and sheep-derived strains in Spanish. In addition, the sequence of T. ovis was clustered into a clade with published sequences of yak-derived strains from Qinghai, and sheep-derived strains from Qinghai, Sudan, Turkey, Egypt, and Xinjiang, while the sequence of T. uilenbergi was clustered into a clade with sequences of Qinghai vole-derived strains, goat-derived strains from Hunan and India, and sheep-derived strains from Iraq and Turkey. Conclusion The piroplasm infection in Tibetan Sheep were serious, with diverse species and haplotypes of the infected piroplasms in some areas from Qinghai Province, and the Theileria sp. OT3 was detected in Tibetan sheep, which provides a scientific support for the comprehensive prevention and control of piroplasmosis in Tibetan sheep in Qinghai.