Horses might play an important role as reservoir hosts in the epidemiology ofEnterocytozoon bieneusi,which is one of the most important zoonotic microsporidian pathogens, with a wide range of hosts. Nevertheless, limited information is available on the infection rates and genotypes ofE. bieneusiin horses, and no data are available on the occurrence and molecular characteristics ofE. bieneusiin horses in Turkey. We determined the prevalence ofE. bieneusiamong horses raised on farms from two provinces of Central Anatolia Region, by amplification of the partial small subunit ribosomal RNA gene using nested PCR. We identified the genotypes ofE. bieneusiisolates by analyzing the ribosomal internal transcribed spacer (ITS) sequences. The overall prevalence ofE. bieneusiwas 18.7% (56/300), with no significant differences in infection rates among age groups or between genders of horses. Sequence analysis revealed eight genotypes: two known genotypes (ERUSS1, BEB6) and six novel genotypes (named ERUH2 to ERUH7). The genotype ERUSS1 was the most common and was found on all farms, age groups, and genders. Phylogenetic analysis clustered all the identified genotypes in ruminant-specific group 2. Our findings contribute to the molecular epidemiology ofE. bieneusi.