To develop an alternative genotyping tool, the genetic diversity of Encephalitozoon hellem was examined at the polar tube protein (PTP) locus. Nucleotide sequence analysis of the PTP gene divided 24 E. hellem isolates into four genotypes, compared to two genotypes identified by analysis of the internal transcribed spacer of the rRNA gene. The four PTP genotypes differed from each other by the copy number of the 60-bp central repeat as well as by point mutations. A simple PCR test was developed to differentiate E. hellem genotypes based on the difference in the size of PTP PCR products, which should facilitate the genotyping of E. hellem in clinical samples.