DNA amplification by PCR and sequence analysis of the 5.8S rRNA and flanking internal transcribed spacer regions was performed on forty-two Trichomonas gallinae isolates. The analysis suggests three different species exists within the T. gallinae morphologic complex. One group demonstrated high homology to T. gallinae GenBank sequences; whereas the second group was more closely related to T. vaginalis (98%) than to T. gallinae (92%). The third group shared a 92% identity with T. vaginalis, T. gallinae, and T. tenax. Sequence analysis of the 18S rRNA and alpha-tubulin genes supports the results. Virus purification was performed on twelve T. gallinae isolates to determine if intracellular RNA viruses exist. Viruses were not detected by RNA extraction, reverse transcriptase-PCR, or by electron microscopy following purification attempts. The hemolytic activity of twenty-two T. gallinae isolates was investigated. Mean hemolytic activity ranged from 3.5% to 53.4% and hemolytic activity was not associated with virulence.