Trypanosoma cruzi, the agent of Chagas disease is associated with a very high clinical and epidemiological pleomorphism. This might be better understood through studies on the evolutionary history of the parasite. We explored here the value of antigen genes for the understanding of the evolution within T. cruzi. We selected 11 genes and 12 loci associated with different functions and considered to be involved in host-parasite interaction (cell adhesion, infection, molecular mimicry). The polymorphism of the respective genes in a sample representative of the diversity of T. cruzi was screened by PCR-RFLP and evolutionary relationships were inferred by phenetic analysis. Our results support the classification of T. cruzi in 2 major lineages and 6 discrete typing units (DTUs). The topology of the PCR-RFLP tree was the one that better fitted with the epidemiological features of the different DTUs: (i) lineage I, being encountered in sylvatic as well as domestic transmission cycles, (ii) IIa/c being associated with a sylvatic transmission cycle and (iii) IIb/d/e being associated with a domestic transmission cycle. Our study also supported the hypothesis that the evolutionary history of T. cruzi has been shaped by a series of hybridization events in the framework of a predominant clonal evolution pattern.