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<i>Cyclospora</i> spp. is a food-borne intestinal protozoan, which is widely distributed in the world and poses the risk of zoonosis. In order to reveal the prevalence of <i>Cyclospora</i> spp. in Holstein cattle in partial areas of the Yunnan Province, 524 fresh fecal samples of Holstein cattle were collected from Dali, Kunming, Chuxiong, and Qujing in Yunnan Province. A nested PCR amplification of the small subunit (SSU) rRNA gene of <i>Cyclospora</i> spp. was carried out, and the products of the nested PCR were further analyzed by restriction fragment length polymorphism (RFLP) using <i>Bsp E</i> Ⅰ. The results of the present study showed that 13 samples were positive for <i>Cyclospora</i> spp., and the total infection rate of <i>Cyclospora</i> sp. was 2.48%. The infection of <i>Cyclospora</i> spp. was detected in Dali, Qujing, and Chuxiong. Chuxiong showed the highest infection rate (5.71%), and infection rate in Dali and Qujing was 2.19% and 3.16%, respectively. Interestingly, the infection of <i>Cyclospora</i> spp. was not detected in Kunming. The infection of <i>Cyclospora</i> spp. showed no significant differences among different regions (<i>p</i> > 0.05). <i>Cyclospora</i> sp. infection was detected in all ages and sexes, but the differences were not significant (<i>p</i> > 0.05). Sequencing and phylogenetic analysis showed that five <i>Cyclospora</i> spp. samples were closely related to the <i>Cyclospora</i> spp. of humans, and the others were closely related to the <i>Cyclospora</i> spp. of bovines. The results of the present study suggested that there was an infection of <i>Cyclospora</i> spp. in Holstein cattle in the Yunnan Province, and the <i>Cyclospora</i> spp. showed a risk of zoonosis. Thus, the prevention and control of <i>Cyclospora</i> spp. should be strengthened in the Yunnan Province, China. The results of this investigation provide data references for the further research of Cyclosporiasis in Holstein cattle in the Yunnan Province.