Correct classification of the insect vector is central to the study of arboviral disease. A simple molecular method for identification of the main vectors of the mosquito-borne viruses, dengue, yellow fever, and Rift Valley fever in Senegal, West Africa, was developed. We present a system in which the five mosquito species (Diptera: Culicidae) responsible for the majority of flaviviral disease transmission in Senegal can be reliably identified using small amounts of DNA coextracted during flaviviral screening procedures, via an easy amplification of the mitochondrial gene cytochrome oxidase c subunit I or II (COI or COII, respectively). We observed that despite very similar morphology, the two cryptic disease vector species Aedes furcifer Edwards and Aedes taylori Edwards are highly divergent at the molecular level. This sequence variation was used as a basis for the development of a polymerase chain reaction-restriction fragment-length polymorphism system for the differentiation of the two species. We also present the first investigation of the phylogeny of the culicine mosquitoes based on all COI and COII sequences currently available. There seems to be very low intraspecific variation in both genes, whereas interspecific variation is high. As a consequence, COI and COII are ideal candidates for the molecular identification of disease vectors to species level, whereas deeper divergences remain equivocal by using these genes. This system provides a new technique for the accurate identification of culicine disease vectors in West Africa and provides a basis for the expansion of such methods into the study of a range of diseases.