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To assess the prevalence and the distribution of viruses associated with rugose wood (RW) disease in Tunisian grapevines, surveys were conducted in the main Tunisian grapevine growing areas. A total of 403 samples were collected including autochthonous and international table and wine grapes, and rootstocks. All samples were analyzed by RT-PCR for the presence of Grapevine virus A (GVA), Grapevine virus B (GVB), Grapevine virus D (GVD), Grapevine virus E (GVE), Grapevine virus F (GVF) and Grapevine rupestris stem pitting-associated virus (GRSPaV), using specific primers. Molecular analysis showed that 80.9% (326 / 403) of the tested samples were infected with at least one virus. GRSPaV was the most widespread virus (51.3%), followed by GVA (47.9%), GVD (31.5%), GVF (22.3%), GVB (17.8%), and finally GVE (7.2%). According to grapevines typology, wine grapes were the most infected (93.9%) vines, followed by table grapes (87.8%) and rootstocks (75.0%). Autochthonous grapevine varieties were the less infected (65.9%). This is the first study on RWassociated viruses in autochthonous grapevines and rootstocks and the first report on the presence of GVE and GVF in Tunisian vines.