Aims To develop a multiplex real-time PCR assay using TaqMan probes for the simultaneous detection and quantification of Tobacco etch virus (TEV), Potato virus Y (PVY) and Tobacco vein banding mosaic virus (TVBMV). Methods and Results Specific primer and probe combinations for TEV and TVBMV were developed from the coat protein region of the viral genome. To detect PVY, a primer and probe combination PVY-Univ F, PVY-Univ R and PVY-Univ P for amplifying the coat protein region of the virus genome was employed. The detection limit of multiplex real-time PCR for these viruses was 10 copies mu l-1 of the standard plasmid. The multiplex reaction was successful in the detection of these three pathogens, with no non-specific amplification and cross-reaction. Conclusions This multiplex real-time PCR provides a rapid, effective, specific and sensitive method for the simultaneous detection and quantification of the three pathogens on infected tobacco plants. Significance and Impact of the Study This multiplex real-time PCR 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.