A nested RT-PCR was developed, that allows the generic detection of a subgroup of genetically related viruses with a distinct evolutionary history within the genus Ampelovirus. Members of this lineage are Grapevine leafroll associated virus-4, -5, -6, -9 and two isolates (GLRaV-De and GLRaV-Pr) that have been recently characterized and represent new species. The method involves a one step RT-PCR for the generic detection of Closteroviridae species using degenerate primers that target the HSP70h gene followed by a nested PCR, which detects all virus-members of the lineage and differentiates them from the other grapevine closteroviruses. The 490 bp nested PCR amplicons, corresponding to a phylogenetically informative region, can be sequenced directly to obtain initial genetic information for their partial characterization and rapid classification. Additional primers were designed and successfully used for the specific detection of GLRaV-4, -5, -6, -Pr and -De on respective single or multiplex nested PCR assays. The application of a ramped annealing thermal profile in the nested PCR allowed all amplifications to run in parallel. The developed detection scheme is proposed as a tool that can be used for the enrichment of sequence information of known and uncharacterized ampeloviruses, classified within this lineage, enabling their selective amplification in mixed Closteroviridae virus infections.