New insights into the genetic structure and variability of grapevine leafroll-associated viruses (GLRaVs) gained through worldwide efforts in the last decade or so, and the production and use of new sets of serological reagents, have provided the solid foundation on which the present revision of the taxonomic structure of the family Closteroviridae, and the genus Ampelovirus in particular, is based. A comparative examination of the amino acid sequence divergence of three taxonomically relevant genes [RNA-dependent RNA polymerase (polymerase), heat shock protein 70 homologue (HSP70h) and coat protein (CP)] disclosed a difference among Grapevine leafroll-associated virus 4 (GLRaV-4), -5, -6 and -9 and a group of more recently described viruses (GLRaV-Pr, GLRaV-De and GLRaV-Car) below the 25% limit recently set by the International Committee on Taxonomy of Viruses (ICTV) as a discriminating criterion for the identification of species in the family Closteroviridae. This, plus the recognition that GLRaV-4, -5, -6 and -9 are serologically related, have similar biological and epidemiological traits, and that these viruses and GLRaV-Pr, GLRaV-De, GLRaV-Car have a genome with the same structure and size, supports the notion that they are all genetically divergent variants of a single species, GLRaV-4. The genus Ampelovirus is split into two subgroups designated I and II in recognition of the wide difference in the size and structure of the genome of the present members. Finally, the establishment of a fourth genus within the family Closteroviridae, comprising the unassigned putative species Grapevine leafroll-associated virus 7 (GLRaV-7), Little cherry virus 1 (LChV-1) and Cordyline virus 1 (CoV-1), is justified based on their molecular and biological characteristics that differ from those of members of the other three genera of the family.