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Serotype identification occupies the central part of foot and mouth disease (FMD) diagnosis workflow and vaccination decision tree. In this study, a reverse transcription-multiplex PCR (RT-mPCR) strategy wherein three assays with unique combinations of serotype specific primers targeting the VP1 region was developed to differentiate FMD virus serotypes O, A and Asia 1 based on differential size of the PCR amplicons on agarose gel. Their diagnostic performance relative to the mPCR assay in use in India was evaluated on 169 clinical samples and 210 cell culture grown virus isolates. The relative diagnostic sensitivity was found to be 99.69%, 98.78% and 99.08% for primer combinations 1, 2 and 3, respectively. These assays proved their worth by detecting serotype in three FMD suspected specimens that went undiagnosed in the existing mPCR and also by identifying multiple serotypes in the same sample. Their detection limits varied from log 10 2 to log 10 4 viral RNA dilution and from 100 to 0.1 TCID 50 virus depending on the serotype. The validated novel mPCR assays show promise to be included in the routine diagnostic tool-box to augment the efficiency of diagnosis of FMD virus serotypes that display extreme genetic diversity and a tendency of transboundary dispersal. • Multiplex PCR (mPCR) to differentiate FMD virus serotypes O, A and Asia 1 developed. • Serotype specific primers designed at VP1 produce differentially sized PCR products. • Relative diagnostic sensitivity were 99.69%, 98.78%, 99.08% compared to mPCR in use. • The assays detected serotype in additional FMD suspected samples and mixed infection. • The validated novel mPCR could be a back-up diagnostic to enhance serotyping efficacy. [ABSTRACT FROM AUTHOR]