The genus Vitivirus in the family Betaflexiviridae includes eleven viruses known to infect grapevine: grapevine vitiviruses A, B, D, E, F, G, H, I, J, L and M (GVA-GVM). Three of these viruses, GVA, GVB and GVD, have been associated with the etiology of rugose wood disease in grapevine and cause agronomically significant losses. The other vitiviruses were more recently discovered and their effects on grapevine are undetermined. To certify grape material for propagation as virus tested, an updated reverse transcription PCR (RT-PCR) assay to detect all known vitiviruses is desirable. To accomplish this, multiple grapevine vitivirus sequences were aligned at the amino acid level to search for conserved motifs. Two highly conserved motifs were found at an ideal distance for RT-PCR detection in the RNA-dependent RNA polymerase region of the replicase protein. The amino acid motifs were back translated to create degenerate primers and used to successfully amplify all eleven grapevine vitiviruses. The RT-PCR primers were used to test a panel of vitivirus-infected vines for inclusivity as well as vines infected with closely related viruses in the Betaflexiviridae family (i.e. grapevine pinot gris virus and grapevine rupestris stem pitting-associated virus) for exclusivity. Broader use of these primers to detect vitiviruses in other plant hosts was investigated. In summary, an end-point RT-PCR assay that detects all the known grapevine vitiviruses and potentially other members of the genus Vitivirus has been developed. The universal assay represents an alternative to individual assays to reduce the work associated with the diagnosis of vitiviruses, including for regulatory purposes.