The zoonotic risk and genetic diversity of Enterocytozoon bieneusi (E. bieneusi) and its infection in wildlife were investigated. A total of 70 fecal samples were collected from the South China Tiger Breeding Research Institute in Meihua Mountains and the Fuzhou Zoo of China in Fujian Province. E. bieneusi was assayed on the basis of the internal transcribed spacer (ITS) regions of the ribosomal RNA (rRNA) gene by nested polymerase chain reaction (PCR) with a complementary metal-oxide-semiconductor (CMOS) sensor. Four positive isolates of E. bieneusi were detected (5.7%) from three sika deer and one species of the Hylobatidae family (gibbons). Multilocus sequence typing (MLST) of ITS indicated two genotypes, namely, BEB6 and Type IV at MS1, MS3, MS4, and MS7. Type IV belongs to Group 1 with zoonotic potential. The amplification efficiency at the MS1, MS3, MS4, and MS7 sites was 50% (2/4). Among the four positive isolates, two positive isolates of sika deer were amplified simultaneously at the four sites. Nucleotide sequence analyses showed 1, 2, 1, and 2 nucleotide variant haplotypes at the MS1, MS3, MS4, and MS7 sites, respectively. In the 1, 2, 1, and 2 genotypes, two multilocus genotypes (MLGs) were formed. The comprehensive measures for determining the genetic diversity of E. bieneusi contribute to preventing or controlling the global spread of E. bieneusi in wildlife.