Over a period of few years, Pepino mosaic virus (PepMV) has become one of the most important viral pathogen in tomato production worldwide. So far, five PepMV genotypes (EU, LP, CH2, US2 and US1) have been detected. A real time reverse transcription polymerase chain reaction (RTPCR) procedure, using the fluorescence dye SYBR Green was developed for a rapid and reliable detection of genetically diverse Pepino mosaic virus isolates. This procedure was used for the detection and identification of PepMV in both Solanum lycopersicum and Nicotiana benthamiana species. The melting temperature (Tm) for members of a particular strain was very similar, with a host effect that did not hinder strain identification. Under optimal reaction conditions, sensitivity of the detection was as low as 100 fg of viral RNA from infected plants. This level of sensitivity indicated that the real time RT-PCR developed in the present study could be used for routine plant health assays.