One of the most important properties used to classify Potato virus Y (PVY) isolates is their ability to induce (PVYN) or not (PVYO) veinal necrosis symptoms on the indicator host plant Nicotiana tabacum cv. Xanthi. As an alternative to biological assays, several serological and molecular detection tools have been developed for PVY detection and characterization and these have evolved as our knowledge of PVY has improved. However, the assays that have been previously published are all based on the use of neutral markers (antigenic determinants, sequence data, recombination sites or restriction enzyme cleavage sites), which are unlinked to the biological property being characterized (e.g. veinal necrosis). Using the recently identified molecular determinants of the tobacco leaf necrosis symptom induced by PVYN isolates, a one-step fluorescent [TaqMan (R)] RT-PCR assay, based on a single nucleotide polymorphism (SNP) linked to the necrosis property of PVY isolates, has been des! igned. This assay reliably detects and distinguishes PVYN and PVYO isolates. The method is simple (leaf soak extraction process, gel-free, no post-PCR manipulations), rapid (96 tests in less than 3 h from plants sampling to diagnostic results), sensitive (threshold in a range of 10(4)-10(5) PVY copies), reliable (correctly assigns 42 PVY isolates in their respective group) and allows co-detection of mixed samples containing close to equivalent PVYN and PVYO quantities. All these characteristics suggest that the newly developed SNP assay could be used to reliably classify PVY isolates, as a substitute for biological assays performed on N. tabacum cv. Xanthi