There is limited information about the innate immunity of triatomines against Trypanosoma rangeli, an infectious, non-pathogenic human parasite, and T. cruzi, the causative agent of Chagas’ disease. This study aimed at addressing this gap by studying the in vitro trypanolytic hemolymph activity from insects not infected by T. rangeli or T. cruzi. Eight triatomine species were examined including Rhodnius prolixus, R. robustus, R. colombiensis, R. pallescens, R. pictipes, Triatoma dimidiata, T. maculata, and Panstrongylus geniculatus. The hemolymph of R. prolixus and R. robustus laboratory colonies demonstrated strong trypanolytic activity during the first 14 hours of parasite incubation with 81% lysis for the T. rangeli C genotype, 90% for the E genotype, 95% for T. cruzi discrete taxonomic unit (DTU) TcII, 94% for TcV, 96% for TcVI, 94% for Tcbat, and 90% for Tcmarinkellei. No lysis activity was detected 14 or 24 hours after parasite incubation with the hemolymph of insects from R. pictipes R. pallescens, P. geniculatus, and T. maculata colonies. Identical results (absence of lysis) were observed using hemolymph from R. colombiensis and T. dimidiata laboratory colonies and specimens captured in sylvatic environments. The hemolymph lytic activity against recently obtained cultures (95% decrease in live parasite count) and long-term T. cruzi TcII ones (96% decrease) was similar. Lytic activity was similar in hemolymph from R. prolixus nymphs, males and females and insects fed on chicken or mouse blood. This is the first comparative study of the trypanolytic activity of hemolymph from different triatomine species against T. rangeli and T. cruzi genotypes. Although the chemical structures of such lysis factors and the mechanisms determining their expression have not been fully determined, their identification furthers our understanding of triatomines’ innate immunity and their role in Trypanosoma transmission.