The main vectors of Chagas disease in Ecuador are Triatoma dimidiata and Rhodnius ecuadoriensis. The latter species occupies domestic and peridomestic habitats, as well as sylvatic ecotopes—particularly associated with Phytelephas aequatorialis palm trees—in the western coastal region of Ecuador. In the southern highlands, however, such palm tree habitats are uncommon, and sylvatic populations of R. ecuadoriensis have not previously been reported to date. This study was carried out in five rural communities in Loja Province in southern Ecuador, where manual triatomine searches were conducted in various sylvatic habitats. A total of 81 squirrel nests (Sciurus stramineus) and >200 bird nests and other habitats were searched. One hundred three R. ecuadoriensis individuals were found in 11 squirrel nests (infestation index = 13.6%, density = 2 bugs per nest searched, crowding = 9.5 bugs per infested nest, colonization index = 72.7% of infested nests with nymphs). No triatomines were found in bird nests or other sylvatic habitats. The presence of sylvatic R. ecuadoriensis in the southern highlands of Ecuador has important implications for the long-term control of Chagas disease in the region because of the possibility of reinfestation of dwellings after insecticide-based control interventions.