AbstractA one‐step reverse transcription–polymerase chain reaction (RT‐PCR) is used to detect two cherry flexiviruses, Cherry green ring mottle virus (CGRMV) and Cherry necrotic rusty mottle virus (CNRMV), in Prunus species. This unit presents procedures for collection of plant samples, preparation of total nucleic acids, viral RNA‐rich or total RNA extracts from plant tissues, and subsequent amplification of the viral targets by one‐step RT‐PCR using a pair of consensus primers. The PCR amplicons are visualized by electrophoresis in a 1% agarose gel containing ethidium bromide in TAE buffer and viewed under ultraviolet light. This procedure is rapid, sensitive, reliable, and cost‐effective and is generally useful on a wide variety of plant/virus systems. The use of a semi‐automatic homogenizer for sample preparation and one‐tube RT‐PCR for virus detection makes this approach ideal for screening large numbers of samples. Curr. Protoc. Microbiol. 6:16C.1.1‐16C.1.9. © 2007 by John Wiley & Sons, Inc.