Objective To understand the prevalence of Blastocystis sp. in dairy cattle and its gene subtype in partial areas of Yunnan Province, thereby to provide information support for the prevention and control of the protozoan infection. Methods Fecal samples were collected from Holstein cows and dairy buffalos in Kunming, Dali, and Baoshan City from June 2019 to August 2020. DNA was extracted from the fecal samples, and the small subunit (SSU) rRNA of Blastocystis sp. was amplified by nested PCR and sequenced. The sequence alignment was performed with BLAST on NCBI database. Phylogenetic trees was constructed using neighbor-joining method of MEGA 6.0. SPSS 20.0 and SAS 9.1 software were used to analyze the infection rates of dairy cattle in different regions, seasons, farming patterns, breeds, genders and growth stages, and those factors with significant difference were further analysised by binary logistic regression. Results A total of 841 fecal samples were collected, of which 65 were SSU rRNA positive with the amplified fragment being 1 100 bp in length. The overall infection rate of Blastocystis sp. in dairy cattle was 7.7% (65/841, 95%CI: 5.92%-9.53%). The infection rates were 12.9% (32/248, 95%CI: 8.73%-17.08%), 8.1% (19/236, 95%CI: 4.58%-11.52%) and 3.9% (14/357, 95%CI: 1.91%~5.94%) in Kunming, Baoshan and Dali, with significant differences between places(χ² = 16.601, P < 0.01); the infection rate in summer, autumn and winter was 9.4% (56/599, 95%CI: 7.86%-37.30%), 8.3% (8/96, 95%CI: 2.80%-13.86%) and 0.7% (1/146, 95%CI: 0-2.02%), respectively, showing significant differences seasonally (χ² = 12.411, P < 0.05); the infection rate in captive farming and field farming was 8.3% (58/702, 95%CI: 6.23%-10.30%) and 5.0% (7/139, 95%CI: 1.40%-8.67%), respectively, with no significant difference (χ² = 1.693, P > 0.05); the infection rate among Holstein cows and dairy buffalos was 8.4% (41/490, 95%CI: 5.92%-10.82%) and 6.8% (24/351, 95%CI: 4.20%-9.48%), respectively, showing no significant difference (χ² = 0.671, P > 0.05); the rate in bull and cow was 11.1% (10/90, 95%CI: 4.62%-17.60%) and 7.3% (55/751, 95%CI: 5.46%-9.19%), repectively, presenting no significant difference (χ² = 1.617, P > 0.05); the rate in different growth stages of pre-weaned calves, post-weaned calves, juvenile cattle and adult cattle was 2/18 (95%CI: 0-25.63%), 26.2% (11/42, 95%CI: 12.89%-39.49%), 15.1% (11/73, 95%CI: 6.86%-23.28%) and 5.8% (41/708, 95%CI: 4.07%-7.51%), respectively, showing significant differences between growing stages(χ² = 29.604, P < 0.01). Binary logistic regression analysis showed that season (P < 0.05) and growth stage (P < 0.01) were independent influencing factor for Blastocystis sp. infection in dairy cattle. The sequence alignment found that the SSU rRNA sequence from one fecal sample was 96.3% homologous to reference sequence GenBank KX618192 (ST3 subtype), thirty-five samples were 97.3%-99.7% homologous to the reference sequences MH104059, KC148207 and MH358363 (ST10 subtype), twenty-three samples were 98.7%-100% homologous to the reference sequence MT453998 and MT453999 (ST12 subtype) and six samples were 98.7%-100% homologous to the reference sequence MH358361, MH358362, KC148205 and KC148206 (ST14 subtype). Phylogenetic tree analysis showed that the SSU rRNA sequences of Blastocystis sp. were clustered at to ST3, ST10, ST12 and ST14 subtype clade, respectively, having the constituent ratio of the subtypes being 1.5% (1/65), 53.9% (35/65), 35.4% (23/65) and 9.2% (6/65), respectively. Conclusion Blastocystis sp. infection are found in dairy cattle in Yunnan Province, and its ST3 and ST12 subtypes may have zoonotic transmission risk, thus, it is imperative to strengthen the control and prevention of the pathogen in the province.