Plant viruses including tobacco mosaic virus (TMV), Cucumber mosaic virus (CMV), Tobacco etch virus (TEV), Potato virus Y (PVY) and Tobacco vein banding mosaic virus (TVBMV) are major viruses infecting tobacco and often caused mixed infection.Virus detection is important for the control of viral disease.Therefore, a multiplex RT-PCR and real-time PCR assay were developed to detect TMV, CMV, TEV, PVY and TVBMV.For the multiplex RT-PCR, five specific primer sets were used for detection of the different viruses by single PCR.While a multiplex real-time PCR assay using TaqMan probes for the simultaneous detection and quantification of TEV, PVY and TVBMV was also developed.Meanwhile, the multiplex reaction was successful in the detection of these three pathogens with no non-specific amplification and cross-reaction.These multiplex detection methods provide the rapid, effective, specific and sensitive methods for the simultaneous detection and quantification of five tobacco virus in tobacco plants.These multiplex detection methods will be useful not only for diagnostic,ecological, epidemiological and pathogenesis studies, but also for investigating host/virus or virus/virus interactions, in particular during mix infection.