Background: Several Tritrichomonas species have been found in mammalian hosts. Among these trichomonads T. foetus is often found in the urogenital tract of cattle and the gastrointestinal tract of the domestic cat, resulting in sexually transmitted bovine trichomonosis and fecal-orally transmitted feline trichomonosis, respectively. The aims of the current study were to molecularly characterize clinical isolates of T. foetus in cattle populations in Wyoming, South Dakota, and Montana of the United States of America and to phylogenetically analyze Tritrichomonas species of mammalian hosts. Results: DNA sequencing of rRNA genes showed over 99% identity of the newly described isolates to other bovine isolates. Further, T. foetus isolates of various mammalian hosts originated in different geographic regions worldwide were clustered into two well-defined clades by phylogenetic analysis of rRNA and cysteine protease 2 genes. Clade I consisted of isolates originated from cattle, pig, and human whereas clade II contained isolates of cat and dog. Conclusion: It is concluded that all mammalian Tritrichomonas spp. apparently belong to T. foetus. Analysis of more sequences is warranted to support this conclusion.