Tritrichomonas foetus is a protozoan parasite that colonizes the reproductive tract of cattle as well as the gastrointestinal tract of cats. Bovine tritrichomonosis is a sexually transmitted disease whereas feline tritrichomonosis is thought to be transmitted by the fecal-oral route. Furthermore, T. foetus is known as an essentially apathogenic commensal located in the nasal cavity of pigs. Transmission of T. foetus between the different hosts has to be considered a realistic scenario that may have important implications for the epidemiology of infections and disease. In our study, we generated whole genome sequencing (WGS) data from bovine, feline and porcine T. foetus strains to investigate the genetic (dis)similarities among these diverse strains. As a reference, we used a previously released draft assembly from a bovine T. foetus strain K isolated from an infected bull in Brazil. In particular, we identified single nucleotide polymorphisms (SNPs) and the insertion-deletion (indel) variations within the genomes of the different strains. Interestingly, only a low degree of polymorphism (68 SNPs and indels) was found between the bovine and the porcine strains in terms of variants with a predicted impact of moderate or high and where one species is homozygous for one allele and the other homozygous for the other allele. Conversely, however, a 964 times higher number of such differences was detected by comparing the feline with either the bovine (65,569) or the porcine (65,615) strain. These data clearly indicated a close phylogenetic relationship between bovine and porcine T. foetus but a remarkable genetic distinctness of these two strains from the feline strain. The latter observation was confirmed by PCR-based sequencing of 20 in silico-selected indel markers and five in silico-selected SNP markers that uniformly demonstrated a relatively distant phylogenetic relationship of three independent feline T. foetus isolates in comparison to the bovine and porcine strains investigated. In summary, our comparative genome sequencing approach provided further insights into the genetic diversity of T. foetus in relation to the different host origins of the parasite. Furthermore, our study identified a large number of SNP- and indel-containing sequences that may be useful molecular markers for future epidemiological studies aimed at the elucidation of the transmission patterns of T. foetus within different host species.