In this study, we evaluate mixed discrete typing units (DTUs) of Trypanosoma cruzi present in 69 patients of Chagas disease and 92 Triatoma infestans collected under the entomological surveillance program after more than 20 years of intervention with insecticide spraying in Chile. Our aim is to identify T. cruzi DTUs still circulating in T. infestans and chronic patients to evaluate their impact on the environmental health in endemic areas of the country. Blood DNA or triatomine DNA was used as DNA template for PCR assays. For genotyping, different T. cruzi stocks were used to generate the DNA probes to determine four parasite DTUs or mixtures infecting each patient or vector by means of hybridization assays. We found different frequencies of T. cruzi DTUs in patients and in T. infestans. Tc I was the most frequent found in T.infestans, but was less frequent in humans. In contrast, in humans Tc V was most frequent but was less frequent in T.infestans. In conclusion, there were significant differences between the T. cruzi DTUs circulating in patients and vectors. We discussed these results in the context of what has being reported in Chile before the vector control, in neighboring countries, and the selection pressures existing for T. cruzi populations within the invertebrate and vertebrate hosts.