Primers were designed for RT-nested PCR amplification of the highly variable 293-nt fragment from the 5′ terminal part of the Grapevine virus A (GVA) replicase gene, specific to South African variants of molecular groups I and II. This new technique, along with RT-PCR for simultaneous amplification of variants of groups I, II and III, as well as cloning of amplicons, single-strand conformation polymorphism (SSCP) analysis of clones and sequencing, were used to investigate the populations of variants infecting 16 local Shiraz grapevines with different Shiraz disease (SD) status. The techniques were also used to study variants in GVA-infected grapevines from Australia and the USA. The Australian grapevines included seven plants of cvs Shiraz and Merlot affected by Australian Shiraz disease (AuSD), and one plant of cv. Crimson Seedless with unknown AuSD status. Grapevines from the USA included plants of cvs Chardonnay, Thompson Seedless and an unknown cultivar. The results confirmed the association of certain genetic variants of group II with SD and showed the common presence of these variants in AuSD-affected grapevines from Australia. Interestingly, a variant of this group was also detected in grapevine cv. Chardonnay from the USA, although the disease has not yet been reported from that country. The study also supports an earlier observation that members of group II, closely related to variant GTR1-2, are not associated with the disease. The variants were found only in SD-free grapevines. Results show that variants of the most divergent group III, which are common in South Africa, are also present in Australia and the USA. These variants are not associated with SD, but frequently occur in mixed infections with members of group II in plants affected by this disease in South Africa.