| Summary: | <i>Enterocytozoon bieneusi</i> can cause severe diarrhea in children and adults. However, in China, there are scant studies on <i>E. bieneusi</i> in diarrheal children and adults, with the exception of prevalence and genotyping data in a small number of cities including Hubei, Shanghai, and Heilongjiang. In this study, 196 fecal samples (<i>n</i> = 132 in Chongqing, <i>n</i> = 44 in Shandong, <i>n</i> = 20 in Hubei) were collected, including 91 from children and 105 from adults. Through microscopic examination, 19 positive samples (11 from children and 8 from adults) were detected. Using PCR examination, the internal transcriptional spacer (ITS) region was utilized by nested PCR to detect and characterize <i>E. bieneusi</i>. Twenty positive samples were detected, including 14 from children (≤11 years of age) and 6 from adults. According to the sequence analysis of ITS data, one known zoonotic (D) and seven novel (CQH5-11) genotypes were identified. This is the first molecular epidemiological study of <i>E. bieneusi</i> in diarrheal patients in different regions of China. Therefore, this study can provide useful information for the molecular epidemiology and control of <i>E. bieneusi</i> infection in humans in the future.
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