Adult Aedes aegypti mosquitoes were collected in Puerto Triunfo, central Colombia, where dengue is endemic, during a six month period. Viral infection within the head of each individual mosquito was identified by an immunofluorescent assay (IFA) using a flavivirus-specific monoclonal antibody. The dengue virus serotype, present in each flavivirus-positive specimen, was then determined in portions of the remaining thorax using IFAs with serotype-specific monoclonal antibodies. Among 2065 female Aedes aegypti collected and tested, twenty-four flavivirus-positive individuals were found (minimum infection rate 11.6%), three identified as dengue type-1 and twenty-one as dengue type-2 virus. This was consistent with the isolation of only these two serotypes of dengue virus from dengue fever patients within this town. No vertical transmission of dengue virus could be detected in 1552 male Aedes aegypti collected. This method is inexpensive, simple, rapid to perform and suitable for use in developing countries to identify and distinguish different serotypes of dengue virus in their vectors during eco-epidemiological investigations.