A reverse transcriptase-polymerase chain reaction (RT-PCR) assay to detect Rift Valley fever (RVF) virus RNA in experimentally infected mosquitoes was developed. The specificity of the assay was evaluated with three other phleboviruses; sandfly fever Sicilian (Sabin), sandfly fever Naples (Sabin) and Punta Toro (MSP 3) viruses. The relative sensitivity of the assay, determined by using RVF virus RNA extracted from serial dilutions of virus culture, was approximately 50 plaque forming units. This sensitivity level was 100-fold higher when a nested PCR procedure was used. When the RT-PCR assay was used with coded samples of intrathoracically-infected and uninfected mosquito, the assay detected the virus in all infected mosquitoes. With this assay, it was possible to detect RVF virus RNA in a single infected mosquito in the background of 10, 25 or 50 uninfected mosquitoes.