This work aimed to investigate the differential expression of proteins from two distinct genetic groups of T. cruzi (´DTUs´, TcI and TcII), isolated from chronically infected individuals displaying the cardiac form of Chagas’ disease. For this purpose, epimastigote forms of the two isolates were cultured in vitro and the cells recovered for protein extraction. Comparative two-dimensional gel electrophoreses were performed and differentially expressed spots selected for identification by mass spectrometry, followed by database searching and protein categorization. The two-dimensional electrophoretic profiles revealed the complex composition of the T. cruzi extracted proteome. In total 46 differentially expressed proteins were identified present in 40 distinct spots found in the comparative gel analyses. The comparative two-dimensional gel electrophoresis allowed detection of major differences in protein expression between two T. cruzi isolates, belonging to the TcI and TcII genotypes. Our findings suggest that patients displaying the cardiac form of the disease harbor parasites capable of exhibiting distinct proteomic profiles.