Wine grapes from two of the most well-established viticultural regions in Greece, Crete and Thira islands, were surveyed to determine the prevalence of the seven major viral diseases included in Greek phytosanitary certification requirements. A total of 14 indigenous grapevine cultivars in 30 commercial vineyards were investigated, amounting to a total of 250 samples. PCR assays were performed for the reliable detection of Grapevine virus A (GVA), Grapevine virus B (GVB), Grapevine leafroll associated virus 1 (GLRaV-1), Grapevine leafroll associated virus 3 (GLRaV-3), Grapevine fleck virus (GFkV), Grapevine fanleaf virus (GFLV) and Arabis mosaic virus (ArMV) in asymptomatic vines. The virological problems of each island were differed. In Cretan grafted cultivars, the most prevalent virus was GVA (the virus was detected in 43% of the samples) followed by GLRaV-3 (21%), GFkV (20%), GLRaV-1 (17%) and GFLV (3%). In self-rooted vines of Thira, 33% of the samples were positive to GVA and none of the other tested viruses was detected. GVB and ArMV were not detected in both islands. Approximately, 40% of the samples were mixed-infected with more than one virus. The production and conservation of virus-free propagation material of the most valuable south Aegean cultivars was accomplished and the evaluation of their agronomical and oenological characteristics of interest is in progress, efforts that could benefit the Greek vineyard.