Objective To evaluate the serological and parasitological status of patients with chronic Chagas disease (CD) after chemotherapy with benzonidazole. Methods Retrospective study of patients treated with benzonidazole (5mg/kg/day for 60days) between 1980 and 2010. Twenty-nine patients who had CD confirmed by two reagent immunological tests and/or one positive xenodiagnosis before treatment were included. Conventional serology (ELISA and IIF) and parasitological tests (haemoculture and N-PCR) were performed. Results At the time of treatment, the mean age of patients was 36±7.24years (20-39years) and the time post-treatment varied from 1 to 29years. After chemotherapy, all individuals had reagent ELISA and 93.1% had positive results for the IIF test. T. cruzi DNA was detected by N-PCR in 48.3%. Negative results were observed in 41.4% and inconclusive ones in 10.3%. Haemoculture was negative for all individuals. Conclusions Our results suggest that N-PCR may be useful in the early identification of therapeutic failure of CD. 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