A real-time reverse-transcription polymerase chain reaction (RT-PCR) was developed for efficient detection of genetically diverse Pepino mosaic virus (PepMV) isolates. The novel detection system was designed to use a duo-primer system targeting the conserved region in the triple gene block 2 (TGB2) gene with a single conserved TaqMan probe to broaden its reaction to cover all available PepMV strains. This duo-primer real-time RT-PCR assay was evaluated against US1, US2, Ch1, Ch2 and 25 field isolates collected from six major commercial tomato greenhouse facilities in U.S. and Canada in 2006. Under optimum reaction conditions, sensitivity of the detection was as low as 100 fg of purified viral RNA. This assay was also evaluated for its efficiency in detecting PepMV in various levels of contaminated seed samples. Using immuno-capture sample preparation, real-time RT-PCR was able to detect PepMV in one infested seed in 1000. This level of sensitivity indicated that the one-step immuno-capture duo-primer TaqMan real-time RT-PCR developed in the present study could be used for routine seed health assays.