Sugarcane is one of the major cash crops in Pakistan which has a prime contribution in the GDP. Average yield is very low due to pathogenic epidemics particularly sugarcane mosaic virus. Molecular biology tools have been adopted to increase the defense mechanism of sugarcane against viral attacks. Reverse transcription polymerase chain reaction (RT-PCR) was developed for the detection of sugarcane mosaic virus (SCMV) from the sugarcane samples collected from all major sugarcane cultivation districts in the Punjab, Pakistan. The segments of SCMV-CP gene were amplified from SCMV infected plants and sequenced. The sequencing results did not show 100% homology with any already reported isolate in the GeneBank data base. The sequences were submitted to the NCBI GeneBank and got the accession number KC200152 and KC249907 to KC249917. VIGS is a powerful tool used in functional genomics. Mechanism of VIGS is referred as post transcriptional gene silencing (PTGS) which involves the introduction of exogenous gene into the host plant as a result small interfering RNAs (siRNA) are produced which make a RISC assembly and the DICER like proteins chop the invading RNA as a defense mechanism. All plants naturally produce siRNAs as a defense against viruses and exogenous genes. If they are triggered as exogenous genes, the endogenous genes are silenced. In the present study, VIGS vector pBINTRA6 derived from TRV was modified by inserting SCMV-CP gene (amplified from infected sugarcane plants), GFP gene (amplified from Nicotiana benthamiana 16c GFP transgenic plant) and ChlI gene (amplified from Nicotiana benthamiana plant) for expression in N. benthamiana and sugarcane. Two SCMV-CP gene segments, 313 and 454 bp were cloned in pBINTRA6 to produce two VIGS constructs SCMV-CP/pBINTRA6-313 and SCMV-CP/pBINTRA6-454. Transient expression of GFP and ChlI genes silencing was studies in N. benthamiana GFP transgenic line 16c. SCMV-CP/pBINTRA6-313 has resulted relatively better silencing of GFP and ChlI genes in N. benthamiana than SCMV-CP/pBINTRA6-454. Two sugarcane cultivars HSF-242 and HSF-245 were selected for VIGS vector transformation of SCMV-CP/pBINTRA6-313 construct based on the results and observations of relatively more silencing of GFP and ChlI genes in N. benthamiana. The transformed (transgenic) plants were confirmed by PCR amplification of GUS reporter gene. Moreover, GUS assay was performed to confirm the transformation of plasmids as integral part of plant genome. Knockdown expression was studied by amplification of SCMV-CP gene by real time PCR. No amplification of SCMV-CP gene was seen in the transgenic sugarcane plants which confirmed the virus induced gene silencing of SCMV in sugarcane. Among the two sugarcane cultivars, HSF-242 demonstrated a strong expression for GUS assay. Bioassay i.e. transfection with virulent aphids (Rhopalosiphum maidis) was done on 4-6 fully developed leaves of transgenic sugarcane plants after shifting and acclimatization in soil pots under greenhouse conditions followed by molecular analyses. Results of bioassay determined that transgenic plants exhibited slightly slow growth and weak chlorotic phenotype when compared with the non-transgenic control plants. Results clearly demonstrated that transgenic plants showed resistance developed against sugarcane mosaic virus as a result of initiation of VIGS mechanism. The developed transgenic plants may be subjected to the biosafety studies and variety development in future studies. Moreover, VIGS optimized in the current study can be helpful for identification of new traits and control of other pathogenic diseases through PTGS which will play a key role in value addition and economy of the country.