The presence of Rupestris stem pitting associated virus (RSPaV) can go unnoticed since symptoms appear only if additional viruses are present. Detection by reverse transcription-polymerase chain reaction (RT-PCR) is possible; however, this assay could be unreliable if the tissue that is being tested has detection-interfering compounds, or if the virus has a low titer. This paper reports on (i) use of a recently developed extraction method and internal control to determine which tissues from field-grown grapevines yield extracts that are reliable for virus detection by RT-PCR, and (ii) a survey for RSPaV of different tissues from the Vitis vinifera varieties Riesling, Chardonnay, Cabernet Franc, Merlot, Sauvignon Blanc, Pinot Noir, and Gamay, as well as from the rootstocks 3309 and Riparia, which were harvested in Ontario, Canada, at different times of the year. Amplifiable extracts were obtained from virtually all bud, shoot tip, seed, and cane samples tested. Detectable amounts of RSPaV were generally found in all tissues of infected plants except young buds collected in the summer. A combination of three single buds from dormant canes, less time-consuming than the preparation of cane shavings, was a reliable source for RSPaV detection.