The diagnosis of animal trypanosomiasis in field samples is currently based on clinical suspicion and less frequently on traditional parasitological methods characterized by their very low sensitivity, especially in the subacute and chronic phase of the infection. Molecular tools such as Polymerase Chain Reaction have been shown to reach the highest sensitivity percentages, but without good resolution for all of the South American circulating species (T. vivax, T. evansi and T. theileri) or with specificity for only one of these species. In this study, a new PCR-RFLP for species-specific diagnosis of Trypanosomiasis is presented. This method displayed good resolution, sensitivity and specificity for differentiation of the three aforementioned species identified from naturally infected Colombian bovines and water buffaloes. In addition, epidemiological and clinical data were also collected and analyzed from the sampled animals and significant associations were identified.