A spot nested RT-PCR assay using degenerate deoxyinosine-containing primers was developed, allowing rapid and simultaneous detection of Closterovirus sequences. Nested PCR amplification increased the specificity and sensitivity of detection. The sensitivity was also increased by a factor of 10 by using in addition to the deoxyinosine (dI)-containing primers, respective homologous primers in which dI was substituted by dG in the region of sequence homology. These homologous primers are shorter, having lower degeneracy and higher amplification efficiency than the dI-containing primers. This method was coupled to a similar nested RT-PCR detection method for Vitivirus and Foveavirus sequences. This permitted multiplex RT-PCR amplification of sequences belonging to the three genera in the same reaction tube and the two subsequent nested PCR amplifications (one for closteroviruses and one for viti- and foveaviruses) to run in parallel. Different primers and amplification parameters (additives and thermocycling conditions) were evaluated and optimised, respectively, in order to amplify efficiently all different templates. These improvements permitted the multiplex detection of fovea- and closteroviruses in petiole and cortical scraping preparations from 23 infected field-grown grapevines throughout the year, with the exception of GLRaV-1 in petioles that was only possible from June onwards. Preliminary results show that this method can detect reliably virus species from three genera in grapevine allowing simple, fast and cost-effective testing of a large number of samples in certification schemes.