Species of the subgenus Trypanosoma (Megatrypanum) have been reported in cattle and other domestic and wild ruminants worldwide. A previous study in Brazil found at least four genotypes infecting cattle (Bos taurus), but only one in water buffalo (Bubalus bubalis). However, the small number of isolates examined from buffalo, all inhabiting nearby areas, has precluded evaluation of their diversity, host associations and geographical structure. To address these questions, we evaluated the genetic diversity and phylogeographical patterns of 25 isolates from water buffalo and 28 from cattle from four separate locations in Brazil and Venezuela. Multigene phylogenetic analyses of ssrRNA, internal transcribed spacer of rDNA (ITSrDNA), 5SrRNA, glycosomal glyceraldehyde 3-phosphate dehydrogenase (gGAPDH), mitochondrial cytochrome b (Cyt b), spliced leader (SL) and cathepsin L-like (CATL) sequences positioned all isolates from sympatric and allopatric buffalo populations into the highly homogeneous genotype TthIA, while the cattle isolates were assigned to three different genotypes, all distinct from TthIA. Polymorphisms in all of these sequences separated the trypanosomes infecting water buffalo, cattle, sheep, antelope and deer, and suggested that they correspond to separate species. Congruent phylogenies inferred with all genes indicated a predominant clonal structure of the genotypes. The multilocus analysis revealed one monophyletic assemblage formed exclusively by trypanosomes of ruminants, which corresponds to the subgenus T. (Megatrypanum). The high degree of host specificity, evidenced by genotypes exclusive to each ruminant species and lack of genotype shared by different host species, suggested that the evolutionary history of trypanosomes of this subgenus was strongly constrained by their ruminant hosts. However, incongruence between ruminant and trypanosome phylogenies did not support host– parasite co-evolution, indicating that host switches have occurred across ruminants followed by divergences, giving rise to new trypanosome genotypes adapted exclusively to one host species.We are grateful to several students and collaborators for their invaluable assistance with the fieldwork. We thank Prof. Erney P. Camargo for helpful comments. We thank Flobert Njiokou (University of Yaoundé I, Cameroon) and Stephane Herder (IRD/CIRAD, Montpellier, France) for DNA samples from African antelopes. This work was supported by grants from the Brazilian agency CNPq within the UNIVERSAL program. Herakles A. Garcia is a recipient of scholarships from CDCH-UCV in Venezuela, Adriana C. Rodrigues is a postdoctoral fellow of PROTAX-CNPq and Antonio H.H. Minervino is a postdoctoral fellow of CNPq