Fecal specimens were obtained from 12-24-month-old dairy heifers on farms in Vermont, New York, Pennsylvania, Maryland, Virginia, North Carolina, and Florida. PCR positive specimens for Enterocytozoon bieneusi were found in 131 of 571 heifers examined (23%) and on all the farms visited. The prevalence of E. bieneusi varied considerably across farms, with the lowest prevalence (4.7%) on MD-2 and the highest prevalence (37.8%) on NY-2. All PCR positive specimens that amplified the ITS region as well as a portion of the flanking large and small subunit ribosomal RNA genes were sequenced to determine the genotype(s) of the E. bieneusi present and six genotypes were identified. Most were identified as cattle-specific genotypes, previously reported from cattle as BEB1, BEB2, BEB3, and BEB4. Two isolates were genetically identical or similar to E. bienesusi reported as the human pathogens Peru 6 and Peru 9 (or D) genotypes. Although our data demonstrate the presence of zoonotic genotypes in cattle, most genotypes found in cattle were host specific.