The increase in outbreaks of Chagas disease in Colombia has required the strengthening of entomological surveillance with the active participation of the affected communities and the monitoring of the natural infection of the collected kissing bugs recollected inside households. The natural infection with Trypanosoma cruzi of triatomines collected by inhabitants of some municipalities of the department of Antioquia in 2019 was evaluated by molecular methods. This study described the intradomiciliary presence of Panstrongylus geniculatus (Latreille, 1811) in four cities of Antioquia: Barbosa, Liborina, Ituango, and Puerto Triunfo. This vector is reported for the first time in the municipalities Liborina, Barbosa, and Ituango. Furthermore, the natural infection with T. cruzi, DTUI, was reported in Barbosa and Liborina. The epidemiological implications of these findings are analyzed within the context of recent reports of outbreaks of Chagas disease in Antioquia.