Trypanosoma vivax and Trypanosoma evansi are livestock parasites of economic importance in Africa, Asia and South America. In the Pantanal, Brazil, they cause economic losses in both cattle and equines. Little is known of their maintenance and spread in nature, particularly in terms of reservoirs and means of mechanical transmission. Here we report for the first time the use of PCR for the detection of T. vivax and T. evansi in bovines, buffaloes and sheep. Whereas parasitological diagnosis detected only two T. vivax infections, one in buffalo and another in a cow, PCR detected infections in 34.8% buffaloes, 44.7% bovines and 37.3% sheep. Trypanozoon primers detected 41.8% infections in buffaloes and 8.1% in cattle. PCR revealed 6.9% mixed infections in buffaloes and 5.3% in cattle. The potential role of cattle and buffaloes as hosts and reservoirs of T. vivax is discussed, as well as the implications of possible extravascular foci in the maintenance of livestock trypanosomosis.