Abstract: Background and Objective: Groundnut Bud Necrosis Virus (GBNV) (family Bunyaviridae , genus Tospovirus) is an emerging plant viral disease. The GBNV was a very broad host range infecting many economically important crops throughout in India. So the aim of this study is to survey, screening, identification of GBNV infecting onion to know the genetic diversity and compare the sensitivity limit of ELISA, RT-PCR and IC-RT-PCR in GBNV infected onion samples. Materials and Methods: The straw colored, mosaic and necrotic lesions exhibiting by young leaves of onion plants were collected from different locations in South India. The disease samples (No. of samples = 73) were initially screened by DAC-ELISA by using the GBNV coat protein polyclonal antibodies. Total RNA was isolated from the positive ELISA samples and amplified with GBNV coat protein gene specific primers. Comparison of the sensitivity limit of ELISA, RT-PCR and IC-RT-PCR in GBNV infected onion samples. Results: In DAC-ELISA, 50 (68.49%) samples were confirmed as GBNV infected from collected onion samples (n = 73) in the field. In RT-PCR, 61 samples (83.56%) were confirmed by RT-PCR method and 68 samples (93.15%) were confirmed by IC-RT-PCR based on the coat protein gene of GBNV. The sequence analysis revealed that the coat protein gene shared 93-100 and 95-100% sequence identity with GBNV at the nucleotide and amino acid levels, respectively. The sensitivity was compared in DAC-ELISA, RT-PCR and IC-RT-PCR showed 10–3, 10–5and 10–6dilutions, respectively and not observed in healthy onion samples. Conclusion: The IC-RT-PCR was found to be more sensitive than RT-PCR and ELISA for the detection of GBNV.