In this study, 338 fecal samples were analyzed for Enterocytozoon bieneusi from cattle farms in Florida, Maryland, Michigan, New York, North Carolina, Pennsylvania, Virginia, and Portugal. The internal transcribed spacer region (392 bp) of the rRNA gene of E. bieneusi was amplified using a nested PCR protocol. Thirty-two E. bieneusi-PCR positive samples were sequenced. A high degree of genetic polymorphism, represented by five distinct genotypes (BEB1-BEB5), was found among the E. bieneusi isolates from cattle. Most of the isolates formed a distinct cluster consisting of only the four cattle genotypes, but six isolates of a genotype clustered together with E. bieneusi genotypes from humans and other domestic animals. Therefore, only some E. bieneusi isolates from cattle may be of public health importance.