An automated one-step real-time reverse transcription polymerase chain reaction (rRT-PCR) protocol was optimised and evaluated for the routine diagnosis of foot-and-mouth disease (FMD). Parallel testing of RNA samples (n = 257) indicated that this assay has a diagnostic sensitivity at least equivalent to the automated two-step rRT-PCR protocol previously used for the laboratory detection of FMD virus (FMDV). This more rapid and economical one-step protocol will play a key role in contingency planning for any future outbreaks of FMD in the United Kingdom (UK).