Mithun (Bos frontalis) is a rare bovine species that is distributed in Yunnan Province, southwestern China. However, the prevalence of that species in mithun is still poorly understood. The prevalence and species of Cryptosporidium spp. in the fecal matters in mithun were examined at 5 locations in Yunnan province, southwestern China. On the basis of the analysis of the 18S rRNA gene by nested PCR (nPCR), followed by DNA sequencing, 39 (6.24%) of 625 fecal specimens collected from 5 locations were positive for Cryptosporidium spp. In different locations, the Cryptosporidium spp. has prevalence rates ranging from 1.27–10.04%, with statistical differences between the five locations (P < 0.05). In four seasons, the Cryptosporidium spp. has prevalence rates ranging from 0 to 15.60%, with obvious differences between the four seasons (P < 0.001). Four Cryptosporidium species were found, known as Cryptosporidium struthionis (n = 18), Cryptosporidium andersoni (n = 10), Cryptosporidium ryanae (n = 1), and Cryptosporidium sp. (n = 10). Until now this is the only report to examine the prevalence and molecular identification of Cryptosporidium spp. in mithun in Yunnan province, southwestern China, which affects public health safety and should be prevented by appropriate measures.