A high-throughput multiplexed assay was developed for the differential laboratory detection of foot-and-mouth disease virus (FMDV) from viruses that cause clinically similar diseases of livestock. This assay simultaneously screens for five RNA and two DNA viruses by using multiplexed reverse transcription-PCR (mRT-PCR) amplification coupled with a microsphere hybridization array and flow-cytometric detection. Two of the 17 primer-probe sets included in this multiplex assay were adopted from previously characterized real-time RT-PCR (rRT-PCR) assays for FMDV. The diagnostic accuracy of the mRT-PCR assay was evaluated using 287 field samples, including 247 samples (213 true-positive samples and 35 true-negative samples) from suspected cases of foot-and-mouth disease collected from 65 countries between 1965 and 2006 and 39 true-negative samples collected from healthy animals. The mRT-PCR assay results were compared to those of two singleplex rRT-PCR assays, using virus isolation with antigen enzyme-linked immunosorbent assays as the reference method. The diagnostic sensitivity of the mRT-PCR assay for FMDV was 93.9% (95% confidence interval [CI], 89.8 to 96.4%), and the sensitivity was 98.1% (95% CI, 95.3 to 99.3%) for the two singleplex rRT-PCR assays used in combination. In addition, the assay could reliably differentiate between FMDV and other vesicular viruses, such as swine vesicular disease virus and vesicular exanthema of swine virus. Interestingly, the mRT-PCR detected parapoxvirus (n = 2) and bovine viral diarrhea virus (n = 2) in clinical samples, demonstrating the screening potential of this mRT-PCR assay to identify viruses in FMDV-negative material not previously recognized by using focused single-target rRT-PCR assays.