Grapevine is an economically most important and major vegetatively propagated fruit crop in the world and infected with several widespread viruses that seriously affect the economic status of this crop. Currently more than 64 grapevine viruses have been reported. Among these, Grapevine leafroll disease (GLD) is considered as the most economically damaging disease in grapevine-growing regions. GLD is the group of eleven viruses that belong to genus Ampelovirus and family Closteroviridae. Some of the viruses transmitted through vectors and some are graft transmissible. Reliable and accurate diagnostic methods are required for the evaluation of Grapevine leafrollassociated viruses (GLRaVs) and for the control and sanitary selection of GLD. In the present study various diagnostic tool were used to screen Grapevine leafrollassociated viruses from the grapevine germplasm of Pakistan. In the three provinces of Pakistan, symptomatic and asymptomatic leaves along with petioles were sampled from 13 vineyards. Leaf curling, reddening and yellowing of leaves were observed in few cultivars while mostly samples were asymptomatic. PCR based method is considered as the most sensitive and accurate for the detection of infectious pathogen at their early infection. For this purpose, RNA extracted from two methods was analyzed for conventional PCR by using specific primer sets that target the conserved regions. Total 85 samples out of 249 were detected for GLRaVs by conventional PCR. A TaqMan RT-PCR is the most significant, sensitive and accurate method in the medium and was also used to analyze the prevalence of infected samples. The extracted RNA quality was checked by using the 18S rRNA TaqMan assay as an RNA specific internal control to prove the better detection methods. The Ct value of 18S was in the range of 3.4-13.03. Two hundred and forty-nine samples were tested for 11 GLRaVs using TaqMan RT-PCR. The most prevalent virus was GLRaV-2 that was detected in 95 samples and showed 38% infection rate. The second most prevalent virus was GLRaV-3 with 7.2% infection rate. GLRaV-4 strain-9 and -Car were negative for all samples. Mixed infections were detected in 40 samples with 16.1 % infection rate. Detection of viruses by TaqMan assay is 10,000 times more sensitive and efficient than the conventional PCR. In the present study, conventional PCR detected 34.1% GLRaVs and RT-qPCR detected 48.2% infection of GLRaVs in the tested samples. This study also provided the superiority of Next-Generation Sequencing (NGS) over the molecular detection assays to identify virome in single grapevine plant. This study analyzed the total RNA sequences by using Illumina Nextseq 500 Platform, ~35000Mb of sequence data were developed from reverse transcribed cDNA and analyzed for sequences of infectious pathogens such as viruses, viroids, fungi and bacteria. The presence of De novo assembly of sequenced reads was identified by BLAST analysis. Total 23 plant viruses, three viroids, two Satellite RNA viruses and one fungal virus were detected in the tested samples. These viruses and viroid belongs to the family Tymoviridae, Closteroviridae, Secoviridae, Betaflexiviridae, Pospiviroidae and Partitiviridae. Genetic diversity of GLRaVs from the infected grapevine varieties of Pakistan was also studied on the basis of nucleotide sequence of full genome and amino acid sequences of the coat protein (CP), RNA dependent RNA polymerase (RdRp) and heat shock protein 70 homologous (HSP70h). The phylogenetic analysis indicated that full genome represent best phylogeny of GLRaVs. Phylogenetic analysis on the base of amino acid sequences showed that CP is the more conservative region as compared to RdRp and HSP70h. The full genome of all GLRaVs except GLRaV-3 and GLRaV-4 strain-Pr showed homology with the isolates of USA. This study first time reports the eradication of Grapevine leafroll-associated viruses by excising apical meristem of 0.5mm of infected vine. TaqMan RT-PCR was used to check the sanitary conditions for the screening of GLRaVs and results showed complete eradication of GLRaVs. The objective of this study was to provide the baseline knowledge about the incidence and prevalence of Grapevine leafroll-associated viruses in Pakistan that helps the growers to make better decisions to clean the vineyards in Pakistan. Overall, this is the first study on the detection of grapevine viruses belongs to the family Closteroviridae in Pakistan.