Ten isolates of Trypanosoma evansi from the Pantanal region of Brazil, recently derived from coati (Nasua nasua, carnivora, Procyonidae), horses and dogs, were characterized on the basis of biological (experimental infections in Wistar rats) and biochemical (multilocus enzyme eletrophoresis) data. Biological data were analyzed by Nested analysis of variance and Kruskal-Wallis. Marked heterogeneity in virulence was observed in the isolates. Some of the isolates showed an undulating parasitaemia, typical for African trypanosomes. This biological heterogeneity did not correspond with the biochemical homogeneity observed in the T. evansi isolates. T. evansi has one of the widest distributions and greatest range of mammalian hosts and is widely recognized to have evolved from Trypanosoma brucei. Adaptability of T. evansi was not reflected in the variability of biochemical and molecular parameters studied to date. The variability in virulence was very significant, but not correlated with the host from which it was derived. These data suggested that, in the region studied, T. evansi is transmitted among both domestic and sylvatic animals in one single transmission cycle.