Enterocytozoon bieneusi, the most common zoonotic pathogen of microsporidiosis, has been found in various animals and humans, but no information is available concerning the prevalence and genotypes of E. bieneusi in white yaks (Bos grunniens). In the present study, 353 faecal samples from white yaks in Tianzhu Tibetan Autonomous County, Gansu Province, Northwestern China, were collected and examined by PCR amplification of the internal transcribed spacer gene to estimate E. bieneusi prevalence and identify their genotypes. Of the 353 faecal samples, 4 (1.13%) were tested E. bieneusi-positive. Sequences analysis revealed that two known genotypes, namely, I (n = 1) and BEB4 (n = 2), and a novel genotype, namely, WCY1 (n = 1), were found in this study. Among them, genotype WCY1 was clustered into Group 1, and genotypes I and BEB4 belonged to Group 2. The present study firstly indicates the existence of E. bieneusi in yaks in Gansu Province, Northwestern China. This is also the first record of E. bieneusi in white yaks. Effective measures should be taken to control E. bieneusi infection in white yaks, other animals, and humans.