Microsporidia are composed of a highly diverse group of single-celled, obligate intracellular fungi that colonize an extremely wide range of other eukaryotes, among which Enterocytozoon bieneusi is the most common species responsible for human microsporidiasis. Genotyping of E. bieneusi based on sequence analysis of the ribosomal internal transcribed spacer (ITS) has recognized ~500 genotypes in humans and a great variety of other mammals and birds. Those genotypes vary in genetic or hereditary characteristics and form 11 genetic groups in phylogenetic analysis of the ITS nucleotide sequences. Some of genotypes in Group 1 (e.g., D, EbpC, and type IV) and Group 2 (e.g., BEB4, BEB6, I, and J) have broad host and geographic ranges, constituting a major risk for zoonotic or cross-species transmission. By contrast, host specificity seems common in Group 3 to Group 11 whose members appear well adapted to specific hosts and thus would have minimal or unknown effects on public health. Multilocus sequence typing using the ITS, three microsatellites MS1, MS3, and MS7, and one minisatellite MS4, and population genetic analysis of Group 1 isolates reveal the occurrence of clonality, potential host adaptation, and population differentiation of E. bieneusi in various hosts. Nonetheless, it is still highly desirable to explore novel genetic markers with enough polymorphisms, to type complex or unstructured E. bieneusi populations of various host species and geographic origins, notably those belonging to Group 2 to Group 11. Additional population genetic and comparative genomic data are needed to elucidate the actual extent of host specificity in E. bieneusi and its potential impacts on zoonotic or interspecies transmission of microsporidiasis.