Trypanosoma cruzi, the causal agent of Chagas disease, is mainly transmitted by insects of the Triatominae subfamily. In Colombia, there are 26 triatomine species, and 16 of them are naturally infected with the parasite. The parasite loads of naturally infected vectors can be significant in targeting specific species that can affect the epidemiology of the disease. Studying their ecology and behavior is vital to understand their role in T. cruzi transmission dynamics. We evaluated the parasite loads of 182 field-collected triatomines corresponding to 10 species in 13 departments across Colombia. We standardized a methodology to quantify T. cruzi DNA in these insects. We obtained a LOD (limit of detection) of 3.05 p-eq/mL. The 82% of triatomines we evaluated were positive for T. cruzi infection, with loads ranging from hundreds to millions of equivalent parasites per milliliter. Panstrongylus geniculatus, Rhodnius prolixus, and Triatoma dimidiata were the species with the highest loads of T. cruzi; however, other species whose role as vectors is still unknown were also found with high loads of parasites. Our results suggest the relevance of secondary species for T. cruzi transmission in Colombia. We hope our data can help improve entomological surveillance and vector control programs in the country and the region.