| Summary: | <i>Enterocytozoon bieneusi</i> is a fungus-like protist that can parasitize in the intestines of humans and various animals causing a threat to public health. However, there has been no data for <i>E. bieneusi</i> prevalence and genotypes in black goats in Yunnan Province, Southwestern China. In this study, 907 fecal samples were collected from black goats in 5 counties from Yunnan Province. The prevalence and genotypes of <i>E. bieneusi</i> were examined by nested PCR amplification targeting the nuclear internal transcribed spacer (ITS). Multi-locus sequence typing (MLST) was used to further examine the potential occurrence of genetic segregation. The overall prevalence of <i>E. bieneusi</i> in black goats in Yunnan Province was 10.3% (93/907). Statistical analysis revealed that <i>E. bieneusi</i> prevalence was significantly associated with the region, age and gender of black goats (<i>p</i> < 0.001). Four new genotypes (CYG-1, CYG-2, CYG-3, CYG-4) and 11 known genotypes (CHG1, CHG2, CHG3, CHG5, CHG28, J, D, BEB6, Wildboar3, CD6, SDD1) of <i>E. bieneusi</i> were identified. At the microsatellite and minisatellite loci, 15, 2, 17, and 33 sequences were obtained, respectively, forming one new multi-locus genotype (MLG27). Phylogenetic analysis revealed that all 15 genotypes were clustered into group 1 and group 2, with zoonotic potential. This is the first report of <i>E. bieneusi</i> prevalence and genotypes in black goats in Yunnan Province, China. Effective control strategies and measures should be taken to reduce the risk of <i>E. bieneusi</i> transmission between black goats, other animals, and humans.
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