The parasite Trypanosoma cruzi (Kinetoplastida, Trypanosomatidae) can be classified based on biochemical and molecular markers, into six lineages or discrete typing units (DTUs), T. cruzi I-VI (TcI-VI), from which TcI and TcII are the parental genotypes. Trying to understand the dispersion of the subpopulations of T. cruzi in nature and its complex transmission cycles, the serine carboxypeptidase genes of T. cruzi were used as a molecular marker in the present study. DTUs of 25 T. cruzi isolates derived from different hosts and from different regions of Brazil were classified. Using specific primers, the complete serine carboxypeptidase open reading frame of 1401 bp was sequenced. The obtained data shows significant differences in the sequences of TcI and TcII. The analysis of the T. cruzi significantly different serine carboxypeptidase genes allowed distinguishing between the parental DTUs TcI to TcII and the hybrid DTU TcVI which grouped within the latter branch. The sequence diversity within the T. cruzi subpopulations was rather low. The analysis using the genes encoding proteases seems to be an interesting approach for the reconstruction of the origin and genotype evolution of T. cruzi.