A region encoding the putative RNA replicase domain of Grapevine fleck virus (GFkV) was amplified from infected grapevine samples using the reverse transcription-polymerase chain reaction (RT-PCR). Two different sizes of virus specific PCR products were consistently detected in different grapevine varieties using the same pair of primers. We selected four reference vines and carried out nucleotide sequence analysis. The size of the small product was 353 nucleotides (nt) and that of the large product was 416 nt. Database search showed that both products shared a high sequence homology with GFkV. Sequence variants producing the small size products were named GFkV353, while those giving the large size products were named GFkV416. The small size product shared a higher sequence homology with that of the published GFkV sequence from Italy, which had the same product size of 353 nt, while the large product had a 63 base insertion in that region. This insertion occurred within domain VI of the replicase gene. A survey was carried out for the presence of these two sequence variants in Australia, New Zealand, the USA, South Africa and a number of other countries. In Australia, of 3874 grapevine samples tested by the RT-PCR assay, 492 (12.7%) contained GFkV353 and 145 (3.7%) contained GFkV416, while only six (0.1%) contained both variants. The sequence homology between these two amplified products and the corresponding regions in related viruses of tymoviruses and marafiviruses is discussed.