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The prevalence and the genetic diversity of grapevine virus D (GVD) isolates from rootstocks, wine and table grape varieties grown in Tunisia were studied. RT-PCR assays performed on the coat protein gene (CP) showed the presence of GVD in 31.5% of the 403 samples tested. The highest rate of infection was found in table grapes (56.5%), followed by autochthonous table grapes (24.1%), wine grapes (20.8%) and rootstocks (12.5%). Sequences and phylogenetic analyses of the partial CP genes of 14 GVD isolates showed nucleotide identities that ranged from 84% to 99%. The Tunisian GVD-isolates segregated in 3 phylogenetic groups together with international isolates reported in GenBank. The present study extends our knowledge of the presence of GVD in Tunisian vines and on its genetic diversity, which is useful for developing broad-spectrum molecular diagnostics (RT-PCR) capable of detecting the different isolates infecting vines.