Thirty-one trypanosomatid stocks were isolated from various sylvatic hosts and vectors in two different regions from the Paraná State of Brazil. The stocks were analyzed by multilocus enzyme electrophoresis (MLEE) on cellulose acetate plates (22 genetic loci). All stocks were unambiguously attributed to Trypanosoma cruzi, and were found to be closely related to the formerly described zymodeme I (TC1 subgroup of T. cruzi). By comparison with other sylvatic cycles with similar sample sizes (Southern USA, Colombia, French Guiana), genetic variability among these stocks was very limited, with only two variable loci out of 22, and only three different multilocus genotypes. Population structure of T. cruzi in these cycles appears to correspond to a set of very closely related clonal genotypes. This very limited genetic variability could be due to a recent foundation of these populations (founder effect). The implications of an active Chagas sylvatic cycle in this area are discussed.