Among approximately 14 human-pathogenic microsporidian species, Enterocytozoon bieneusi is the most common. It can inhabit the small intestines, causing chronic diarrhea and wasting syndrome. Prevalence and genotype data for E. bieneusi in humans is available for only a few provinces of China. In the current study, 93 fecal specimens were collected from diarrheic children in Chongqing. Polymerase chain reaction amplification and sequencing of the internal transcribed spacer (ITS) region of the E. bieneusi rDNA sequence identified 11 (11.83%) positive specimens. Among them, 8 (8.60%) are from patients of ages ranging from 2 mo to 6 yr old and 3 (3.23%) from patients 7 to 11 yr old. In total, 6 genotypes (4 novel genotypes and 2 known genotypes) were identified in this study. Phylogenetic analysis showed that all the genotypes identified in the present study belong to group 1, which previously has been described as a zoonotic group. This could mean these infections were acquired zoonotically, and it may be prudent to warn those people having close contact with animals of this potential risk.