Dengue is the most important arboviral disease worldwide. Dengue diagnosis is usually made by serology, but serological techniques do not identify the infecting strain, and are only useful late in the course of infection. Several reverse transcription-polymerase chain reaction (RT-PCR) protocols have been described for dengue diagnosis but none of them has been used on a regular basis. We conducted a validation study of PCR-based diagnosis in an area (in Brazil) where dengue-1 virus has been circulating at a low incidence rate. Viral detection by RT-PCR was evaluated using the sera of 253 patients with clinical diagnosis of dengue, and the results were compared to those obtained by IgM capture enzyme-linked immunosorbent assay (MAC-ELISA) and virus isolation. Out of 75 IgM-positive samples, 17 were RT-PCR positive, and only 2 were positive for virus isolation. Through enzymatic digestion of PCR amplicons, we were able to differentiate the 2 dengue serotypes circulating in Brazil (dengue-1 and dengue-2), and to determine that dengue-1 was the virus responsible for the infections. We show with this study that RT-PCR is more sensitive than virus isolation on clinical samples and allows for a rapid detection of dengue infections and for a straightforward identification of the circulating serotype.