A Potato virus Y (PVY) single nucleotide polymorphism (A/G2213), recently identified as a molecular determinant of the tobacco leaf necrosis symptom induced by PVYN isolates, has been used as a target to develop two PVY group-specific (PVYN and PVYO) fluorescent (TaqMan®-based) real-time RT-PCR assays. These procedures allow detection, characterisation, and quantitation of a wide range of PVY isolates in samples containing 103-108 viral transcripts. Moreover, the high specificity of these two new assays make the simultaneous detection and the reliable quantitation of PVYN and PVYO isolates in mixed solutions, regardless of the YN/YO ratio, feasible. The high sensitivity (threshold of 103 copies per reaction) and the PVY group specificity of these two new PVY detection tools clearly improve previously published PVY detection tests and offer new opportunities for PVY research programs.