Multiplex reverse-transcription polymerase chain reaction (mRT-PCR) assay is a sensitive and rapid method for the detection and serotyping of foot and mouth disease virus (FMDV). However, the method has not been used to its full potential, because of factors such as cost, a lack of infrastructure and the complexity of the reaction mixture. This study was undertaken to optimise and validate a thermostable, lyophilised, ready-to-use mRT-PCR kit for the rapid detection of FMDV in field laboratories in India. Trehalose, PEG-8000 and glycerol were evaluated for stabilisation of the PCR mixture at ambient temperatures. The lyophilised mRT-PCR kit was validated and found robust enough for use in field-level laboratories. The PCR reaction mixture in the ready-to-use kit has low complexity, so chances of cross-contamination during the preparation of the mixture are limited, but may easily be monitored by using lyophilised internal positive and negative controls. In addition, the requirement to maintain live FMDV isolates as internal positive controls at field-level regional laboratories is eliminated.