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Methods to determine blood-meal sources of hematophagous Triatominae bugs (Chagas disease vectors) are serological or based on PCR employing species-specific primers or heteroduplex analysis, but these are expensive, inaccurate, or problematic when the insect has fed on more than one species. To solve those problems, we developed a technique based on HRM analysis of the mitochondrial gene cytochrome B (Cyt b). This technique recognized 14 species involved in several ecoepidemiological cycles of the transmission of Trypanosoma cruzi and it was suitable with DNA extracted from intestinal content and feces 30 days after feeding, revealing a resolution power that can display mixed feedings. Field samples were analyzed showing blood meal sources corresponding to domestic, peridomiciliary and sylvatic cycles. The technique only requires a single pair of primers that amplify the Cyt b gene in vertebrates and no other standardization, making it quick, easy, relatively inexpensive, and highly accurate. Author Summary: Chagas disease is one of the most important tropical diseases in America. This disease is caused by the parasite Trypanosoma cruzi and transmitted through the feces of blood-sucking insects known as triatomines. Different species of insects have different habits and food sources that confer variable degrees of epidemiological importance. In this paper, we propose the use of High Resolution Melting (HRM) analysis of cytochrome b (cyt b) gene PCR products to identify blood-food sources in triatomines. This tool can effectively differentiate blood-meal sources of insects collected from the field. Such data allows for targeted investigations of insect species that are likely to be involved in the transmission of the parasite to humans in different regions. This knowledge is very important because it allows establishing and designing vector control and surveillance strategies according to each geographical area and to stop the transmission of the parasite to human populations by insects. [ABSTRACT FROM AUTHOR]