In Chile, the main vector of Chagas disease, Triatoma infestans, is under control after insecticide spraying. However, it has been found colonizing wild habitats. This study evaluated Trypanosoma cruzi infection of sylvatic and domiciliary T. infestans and identified their parasite genotypes. The sample studied was composed mainly of T. infestans sylvatic nymphs and domiciliary adults from a semi-urban area with human dwellings under vector control surveillance. Results showed prevalences of 57.7% in nymphs and 68.6% in adults. Hybridization tests showed a major T. cruzi lineage (TcI) circulating in sylvatic (93.3%) and domiciliary (100%) T. infestans. TcII, TcV, and TcVI were also detected, mainly in nymphs, suggesting differential adaptation of T. cruzi lineages among instars. We also discuss the origin of domiciliary individuals of T. infestans and the risk of human infection by triatomines of sylvatic foci that invade houses despite vector control programs.