A study of the evolutionary relationships of GLRaV-4,-5,-6 and -9, and two new Ampelovirus isolates (GLRaV-Pr and -De) related to grapevine leafroll disease was conducted based on molecular variability, positive selection analysis and maximum likelihood phylogenetic reconstructions. Sequences corresponding to the N-terminal HSP70h and full CP encoding genes were determined for these viruses and datasets including homologous genomic regions from different members of the Closteroviridae were analyzed. GLRaV-Pr and -De were further characterised as distinct from the other closely related species after determination of a large genomic region (4319-4358 nts). ML phylogenetic topologies for both genes established the closer phylogenetic relationships of GLRaV-4,-5,-6,-9,-Pr and -De in regard to the other ampeloviruses, revealing very low inter-species evolutionary distances for this multitudinous lineage. The HSP70h segment phylogeny and bootstrap analysis enabled the identification of species within this lineage and provides a useful taxonomic tool for the rapid demarcation of these viruses. Estimations of d(N)/d(S) using the CP and HSP70h datasets revealed that, within the Closteroviridae, these viruses are subjected to the strongest constraints against amino acid substitutions. These estimations demonstrated a distinct evolutionary trait for this lineage probably related to its particular ecological niche that involves successful adaptation to the host, transmission through vegetative propagation and lack of vectors with high transmission efficiency.