The occurrence of Grapevine fanleaf virus (GFLV) in 74 vineyards in grapevine-growing areas of Andalusia, southern Spain, was investigated, as well as the genetic variability of the coat protein (CP) gene on RNA2. Overall, the prevalence of GFLV (the percentage of vineyards with GFLV) was 24·3%, and it was most common in the Jerez-Xérès-Sherry and Manzanilla-Sanlúcar de Barrameda denomination of origin (DO) (29·4%) and Condado de Huelva DO (24·0%), followed by the Montilla-Moriles DO (13·3%). GFLV-infected plants showed fanleaf and yellow mosaic symptoms together with general vine decline. In almost all samples, except one from Jerez-Xérès-Sherry and Manzanilla-Sanlúcar de Barrameda DO and one from Condado de Huelva DO, GFLV-infected plants showed soil infestation by the virus vector nematode species Xiphinema index and/or the potential vector X. italiae. Reverse transcription-polymerase chain reaction (RT-PCR) successfully amplified the partial CP gene product of the expected size (555 bp) in leaves and nematode vectors. A total of 135 clones of the partial GFLV CP gene were analysed for sequence variation using single-strand conformation polymorphism, yielding 12 different haplotype patterns. Haplotypes were separated among the three different DO. The haplotype patterns MOB, MOC, MMA, MMC and HB were detected in leaf samples and nematode vectors for each locality, except for Jerez de la Frontera-429. Sequence analysis of the GFLV haplotypes revealed sequence variability within the haplotypes and greater abundance of some variants than others in the same sequenced haplotype pattern. Consequently, GFLV in southern Spain exists as a ‘quasispecies’, as it has been reported in other grapevine-growing areas of the world. This paper is the first report on the molecular variability of GFLV in Spain.