Background: Transcriptome data obtained from plant samples often contain significant numbers of reads originating from viral genomes, which are typically co-isolated during the RNA extraction process. This occurs through the simultaneous presence of viral RNA alongside host plant RNA, leading to the inclusion of viral sequences in the transcriptomic data. Methods and results: Here, we identify a novel member of the genus Ampelovirus, grapevine leafroll-associated virus S (GLRaV-S) by employing the high-throughput sequencing (HTS) of RNA of grape leaves showing leafroll symptoms using a bioinformatic pipeline for plant virus detection. The genomic RNA of GLRaV-S, measured 13,102 nucleotides (nts) and encompasses five open reading frames (ORF). Homology analysis of GLRaV-S genome showed sequence identity of 23.0 – 53.3 % with the sequences of known ampeloviruses. Phylogenetic analysis based on genome sequences showed that GLRaV-S clustered in a same clade of ampeloviruses. However, the RdRp and HSP70h of GLRaV-S clustered with subgroup II while the CP sequences clustered with subgroup I of ampeloviruses. Conclusions: Based on the species demarcation criteria, GLRaV-S represents a newly discovered species within the genus Ampelovirus of the Closteroviridae family. This study on identification of novel virus will be useful in developing a robust certification program for the production of healthy plants of grapevine.