The aim of the present work was to investigate the molecular identification and characterization of the three different viruses including Grapevine Fanleaf virus (GFLV), Arabis Mosaic virus (ArMV) and Tobacco ringspot virus (TRSV) from Nepovirus group in the main grape producing regions of Pakistan by using reverse transcription polymerase reaction (RT-PCR) and real time polymerase reaction (qRT-PCR). Total 370 symptomatic and asymptomatic samples were screened for these viruses. Total RNA of all samples was extracted by guanidine isothiocyanate and CTAB method and the efficient one was further used as a template for the amplification by RT-PCR. 28 out of 370 were detected as positive for GFLV while none of other viruses were found positive in the screened samples. The results from the analysis of the conventional PCR confirmed the presence of only GFLV as the gene fragment was amplified by PCR at desired size of 322 bp. One of the positive PCR amplified product of GFLV was subjected to Sanger sequencing and phylogenetic relationship using MEGA 7.0. The phylogenetic analysis showed 90% homology with American isolates. The real-time TaqMan® RT-PCR assay was compared to the conventional RT-PCR assay for the detection of viruses using purified total RNA. This study showed that TaqMan® RT-PCR was more sensitive than conventional RT-PCR for testing different isolates of these viruses present in low titer and it also gave the amount of virus present in each sample. The current study produces a significant preliminary data showing the current status of Nepoviruses that can be used for the establishment of a pathogen tested grapevine germplasm program for grape cultivation in Pakistan.