Thus far, genotyping of Enterocytozoon bieneusi has been based solely on DNA sequence analysis of the internal transcribed spacer (ITS) of the rRNA gene. Both host-adapted and zoonotic (human-pathogenic) genotypes of E. bieneusi have been identified. In this study, we searched for microsatellite and minisatellite sequences in the whole-genome sequence database of E. bieneusi isolate H348. Seven potential targets (MS1 to MS7) were identified. Testing of the seven targets by PCR using two human-pathogenic E. bieneusi genotypes (A and Peru10) led to the selection of four targets (MS1, MS3, MS4, and MS7). Further analysis of the four loci with an additional 24 specimens of both host-adapted and zoonotic E. bieneusi genotypes indicated that most host-adapted genotypes were not amplified by PCR targeting these loci. In contrast, 10 or 11 of the 13 specimens of the zoonotic genotypes were amplified by PCR at each locus. Altogether, 12, 8, 7, and 11 genotypes of were identified at MS1, MS3, MS4, and MS7, respectively. Phylogenetic analysis of the nucleotide sequences obtained produced a genetic relationship that was similar to the one at the ITS locus, with the formation of a large group of zoonotic genotypes that included most E. bieneusi genotypes in humans. Thus, a multilocus sequence typing tool was developed for high-resolution genotyping of E. bieneusi. Data obtained in the study should also have implications for understanding the taxonomy of Enterocytozoon spp., the public health significance of E. bieneusi in animals, and the sources of human E. bieneusi infections.