Some rootstocks, particularly those from American Vitis spp. hybrids, are symptomless carriers with a low concentration of GLRaV-2; the virus can accumulate to high levels in Vitis vinifera scions upon grafting on such roostocks. It is important to increase the sensitivity and to improve the polyvalence of GLRaV-2 detection assays in order to develop reliable detection procedures for the certification of initial grapevine material worldwide. This paper describes the development of polyvalent primers and their use to develop a broad-spectrum reactive and highly sensitive SYBR Green real-time RT-PCR assay (SG qRT-PCR) for the quantitative and polyvalent detection of all the known variants of GLRaV-2.