Grapevine rupestris stem pitting associated virus (GRSPaV) is a component of the rugose wood disease complex. The vast majority of collected samples through field surveys in Californian vineyards that showing rugose wood disease complex symptoms, were tested negative for viruses typically associated with graft union disorders, meanwhile 50% were found positive for GRSPaV using RT-PCR methodology. The dsRNA extracted from GRSPaV-positively tested samples were used as a template for cDNA construction. Molecular analysis of the several sequenced clones from different vines, using ClustalW bioinformatics program revealing significant variations in the virus genome. Two different GRSPaV-isolates named later as GRSPaV-SY and GRSPaV-PN had been identified, showing significant nucleotide sequence variability reached up to 29%. Specific pairs of primers were designed and optimized to detect each isolate specifically, using one step RT-PCR techniques. The use of these detection primers in studying the etiology of GRSPaV and correlation of the symptoms variation will be inevitable.