Antioxidant defense in Trypanosomatids has been indicated as a potential target for chemotherapy. Tryparedoxin peroxidase (TXNPx) participates in this defense by metabolizing hydrogen peroxide to water molecules. In this work, genes encoding both cytosolic (cTcTXNPx) and mitochondrial (mTcTXNPx) TXNPx were characterized in 15 benznidazole-susceptible and resistant Trypanosoma cruzi strains. Northern blot and real-time RT-PCR analyses revealed that the levels of cTcTXNPx and mTcTXNPx mRNA were two-fold higher in the in-vitro-induced resistant 17 LER T. cruzi population than its drug-susceptible counterpart 17 WTS. The mRNA levels for both genes were similar among the other T. cruzi samples studied. No amplification of these genes was observed in the parasite genome. In silico analyses indicated that cTcTXNPx and mTcTXNPx genes present eight and two copies, respectively, dispersed in the parasite genome. By western blot analysis, anti-cTcTXNPx and anti-mTcTXNPx polyclonal antibodies recognized a 23- and 25-kDa peptide, respectively, in all T. cruzi samples analyzed. The expression levels of these native proteins were similar for all samples except 17 LER, which displayed two-fold greater expression. In addition, the oxidized mTcTXNPx protein (50 kDa) demonstrated 5.5-fold greater expression in the 17 LER population than 17 WTS. Our findings demonstrate increased expression of the cytosolic and mitochondrial TcTXNPx in the T. cruzi population with in-vitro-induced resistance to benznidazole.