Eighteen strains of Trypanosoma cruzi isolated from two species of triatomines in the state of Paraná, Brazil, were characterized molecularly using three strategies: RAPD (randomly amplified polymorphic DNA) with four primers, analysis of the D7 polymorphic region of the 24Sα rDNA, and RFLP (restriction fragment length polymorphism) of region 5′ of the mitochondrial gene COII (cytochrome oxidase subunit 2). The phenogram constructed with the RAPD data showed that only three strains isolated from Panstrongylus megistus collected in the Municipality of Arapongas were grouped together in a sub-branch. None of the other 15 strains could be clustered according to triatomine species or geographical origin. The strains were grouped with the T. cruzi I reference sample, indicating closer association with the sylvatic transmission cycle of T. cruzi in the state of Paraná. However, analyses of the rDNA and COII gene polymorphisms revealed the presence of populations from both T. cruzi I and II major lineages. In half of the analyzed triatomines, we found parasites from both lineages coinfecting the same bugs. Of these, most (6/9) were isolated from Triatoma sordida, and 3/9 from Panstrongylus megistus. These results contribute to a better comprehension of the ecoepidemiology of Chagas' disease in Paraná, and raise questions about the role of studies of polyclonal population dynamics for controlling the transmission of T. cruzi to humans in this region.