Epimastigote culture forms of different isolates of Trypanosoma cruzi from different mammal hosts, humans, and vectors were tested with FITC-conjugated peanut agglutinin lectin (PNA–FITC). The parasites maintained in axenic medium, liver infusion tryptose, were evaluated by flow cytometric analyses; whereas T. cruzi I (TcI), which is associated with the sylvatic transmission cycle, was labeled in high percentages with PNA (88–99.2%), T. cruzi II (TcII) (parasites associated with domiciliar cycle) and T. cruzi, zymodeme 3 (Tc/Z3) (also associated with the sylvatic cycle) were labeled in low percentages (TcII, 0–26% and Tc/Z3, 0–12.6%). It was demonstrated that it is possible to differentiate the 2 main T. cruzi subpopulations, TcI and TcII, using Arachis hypogaea. These results also showed a higher variability in TcII in terms of PNA binding.