Chilean T. cruzi populations from different endemic areas and transmission cycles were characterized at several biochemical levels, to mention: hybridization with kinetoplast DNA probes, molecular karyotype, isoenzyme studies and kinetoplast DNA restriction fragment length polymorphism. Furthermore, an immunological approach with immune sera from Balb/c infected mice with different T. cruzi populations was used to differentiate among parasite types by the in vitro complement-mediated lysis assay. Parasite grouping by the above described methods allows to classify T. cruzi populations on a very defined number, suggesting that they have a clonal structure.