Histomonas meleagridis, the causative agent of histomoniasis, is a protozoan parasite classified in the Dientamoebidae (order Tritrichomonadida). The α-tubulin gene of 7 H. meleagridis isolates originating from either domestic chickens or turkeys from the United States was amplified by nested PCR and sequenced. A 91.4–99.8% nucleotide identity was shared among the 7 different sequences, and phylogenetic analysis disclosed that the 7 isolates were divided into at least 3 clades. These sequences had a 91–99% nucleotide identity and a 96–100% amino acid identity compared with 3 H. meleagridis α-tubulin sequences obtained from isolates originating from turkeys in Germany. Further α-tubulin gene analysis from species in the Dientamoebidae will be useful in elucidating the evolutionary relationship of these protozoans.