Extracted DNA from twenty-eight Histomonas meleagridis-infected avian tissue samples from multiple hosts and geographic locations was analyzed for variation in 5.8S rRNA and flanking internal transcribed spacer regions (ITS1 and ITS2) and the 5.8S. Samples were amplified by polymerase chain reaction (PCR), sequenced, and compared with known sequences from GenBank accessions of H. meleagridis and other related protozoa. The analyses revealed significant genetic variation within H. meleagridis sequences suggesting the possibility of multiple genotypes or possible misdiagnosis. Related protozoa found in some samples were identified as Tetratrichomonas spp. A phylogenetic tree analyzing the 5.8S and flanking ITS regions was inconclusive. In contrast, a tree constructed only on the 5.8S rRNA, grouped all but one H. meleagridis sample into one clade, including GenBank accessions from Europe. This suggests that the 5.8S region is more reliable in identifying genera. No correlation between genotypes and host species or geographic location was observed.