Enterocytozoon bieneusi is an obligate intracellular parasite and the most common pathogen of microsporidiosis in humans and animals. In this study, a total of 198 fecal samples were collected from 11 captive populations of Rhesus macaque in Chinese zoos, to investigate the prevalence and analyze the zoonotic potential of E. bieneusi by genotype of the Internal Transcribed Spacer (ITS) gene on the rRNA of E. bieneusi via nested PCR. Results showed that the average infection rate of E. bieneusi in the 11 populations was 13.6%, and the highest infection rate was 56.5% in the population of Xinjiang Tianshan Zoo. Seven genotypes were identified including 2 known genotypes (D and CM1) and 5 novel genotypes (Mul1, Mul2, Mul3, Mul4 and Mul5). Phylogenetic analysis revealed that the novel genotypes Mul2, Mul3, Mul4 and Mul5 belonged to Group 1 showed the zoonotic potential．These findings extend the distribution of E. bieneusi genotypes and provide baseline data for controlling E. bieneusi infection.