<p>Grapevine virus L (GVL) is a newly identified virus that infects grapevines. In this study, twelve GVL isolates from China were identified and subjected to phylogenetic analysis. Based on this, an efficient and rapid real-time fluorescence quantitative RT-PCR (RT-qPCR) assay was developed. Preliminary investigation of 221 grapevine samples collected from the material preservation nursery of the National Center for Eliminating Viruses from Deciduous Fruit Tree by RT-PCR revealed that 5.4% of the tested samples were infected with GVL. The complete genome sequences of twelve GVL isolates were further obtained using RT-PCR and cloning. These GVL sequences shared nucleotide identities ranging from 73.7% to 99.3% among themselves and from 72.6% to 99.4% with previously published GVL isolates in the NCBI database. Phylogenetic analysis based on different ORFs and the complete genome sequences classified the variants into two of the five known phylogroups. The established RT-qPCR assay can detect GVL isolates from two variant groups with a strong linear correlation (R² = 0.998) and a high amplification efficiency (E = 99.0%) in the standard curve. This method was 100 times more sensitive than conventional RT-PCR and can specifically detect GVL. Intra- and inter-group reproducibility tests confirmed the assay’s reliability and consistency. RT-qPCR can be used to detect GVL in a wide range of grapevine sample types. The RT-qPCR detection rates (87%) in 193 samples from different seasons and positions of the material preservation nursery were higher than conventional RT-PCR (70.9%). Additionally, testing 60 field grapevine samples from 15 cultivars across various regions of Liaoning province showed GVL detection rates of 13.3% by conventional RT-PCR and 15% by RT-qPCR, with GVL identified in three grapevine cultivars.</p>