BACKGROUND: Enterocytozoon bieneusi is the most frequently detected microsporidian species in humans, wildlife and domestic animals. In northern China, to the best of our knowledge, no information on E. bieneusi infection has been reported in wild animals. The aim of the present study was to survey the occurrence of and genetically characterize E. bieneusi from a broad spectrum of vertebrate species in this region. METHODS: A total of 1372 small intestine or fecal specimens were collected from 1019 mammals, 121 reptiles and 232 birds in Xinjiang Uygur Autonomous Region (XUAR) and Inner Mongolia Autonomous Region (IMAR), northern China. Each animal species was identified according to morphological characteristics and amplification of mitochondrial genes. Genotype analysis of E. bieneusi was performed by amplifying the internal transcribed spacer (ITS) region. RESULTS: A total of 68 wild animal species were identified, including 34 mammal species, six reptile species and 28 bird species. The average rate of infection with E. bieneusi was 9.7% (133/1372 specimens). Twelve genotypes of E. bieneusi, including BEB6, CHG7, D, E, EbpD, horse1, MWC_d1, NCF2, NCF6, PL14, SN45 and XJHT4, were identified in specimens from XUAR, IMAR and Kyrgyzstan. Phylogenetically, these genotypes belonged to four groups, namely Group 1, Group 2, Group 12 and Group 14. CONCLUSIONS: To our knowledge, this study reports for the first time E. bieneusi genotype NCF2 in marbled polecats (Vormela peregusna), genotype NCF6 in red foxes (Vulpes vulpes), genotype D in grey wolf (Canis lupus), genotypes CHG7, horse1 and PL14 in rodents and genotypes MWC_d1, PL14 and SN45 in wild birds. The results also indicate that genotypes horse1, NCF2 and NCF6 were acquired either by the fecal-oral transmission route or during predator-prey interaction.