A reverse transcription-polymerase chain reaction (RT-PCR) was assessed in laboratory tests to detect the presence of singleAedes aegypti (L.) orEretmapodites quinquevittatus Theobald mosquitoes infected with Rift Valley fever virus in pools of mosquitoes, 50–600 in size, from laboratory colonies or mixed field collections. The viral RNA was detected in all pools containing infected mosquitoes and was shown to be as sensitive as infant mice but more sensitive than Vero cell cultures for virus detection. Pools diluted down to the equivalent of 1:16 000 mosquitoes were also positive by RT-PCR. RNAs from 4 other phleboviruses were negative, there were no false positives and the procedure followed, with the 2 particular primers chosen, gave consistently clear bands of the PCR products on agarose gels without nested PCR being necessary.