The incidence of Rupestris stem-pitting-associated virus (RSPaV) in two vineyards in South Australia was studied by comparing symptoms with the results obtained from biological indexing and from virus detection by reverse-transcription-polymerase chain reaction (RT-PCR). Vineyard 1 was an experimental block comprising a number of varieties grafted onto Vitis rupestris cv. St George rootstocks for the detection of Rupestris stem pitting disease. No swelling at graft union or decline was observed at 3 years, but some vines showed pitting on the rootstock trunk. Vineyard 2 contained Vitis vinifera cv. Shiraz (Syn. Syrah) vines on Paulsen 1103 rootstock, showing a range of symptoms including swelling of the graft union, pitting on the rootstock, leaf reddening and vine decline, resembling those in Syrah Decline, a disorder known to occur in this variety. RT-PCR using the coat-protein-specific primers detected RSPaV in all samples from either vineyard including symptomless V. rupestris cv. St George, which is used as the biological indicator for the virus. In contrast, a pair of primers designed from the replicase gene detected the virus only in symptomatic vines, whereas the symptomless St George control and nonsymptomatic vines in both vineyards tested negative. An assay for 12 other major grapevine viruses showed that none were associated with either type of symptom. Comparison of a 628-bp amplicon on the replicase gene in 13 RSPaV isolates showed that while they had 96–99% nucleotide sequence identity to each other, their identity to the American isolates from California and New York was around 65%. This low homology may indicate that a different virus species is present. The variability was more pronounced between the two vineyards than among the virus isolates in the Shiraz vines within the same vineyard. However, even the vines of the same vineyard did not contain viruses with exactly the same sequence homology. We found no association between the type of symptom expressed and the sequence variation in the amplicons in either vineyard. The sequence variants in Vineyard 1 showed a closer cluster than in Vineyard 2.