The overexpression of non-expressor of PR proteins (NPR1) gene has proved effective in providing resistance to a broad spectrum of pathogens in different plant species, indicating its functionality across a wide taxonomic range. In the present study, NPR1 gene from mustard (Brassica napus) was cloned by using gene specific primers. The gene encoding NPR1 protein was amplified and cloned in pTZ57R/T vector, sequenced and analyzed in silico. Cloned NPR1 gene had 98 per cent homology with reported NPR1 gene of mustard at both nucleotide and protein level. It has four exons with three stretches of internal introns (642-851,1588-1801,1970-2055). The NPR1 protein of mustard has an ankyrin repeat and a BTB/POZ (Broad-complex, Tramtrack, and Bric-a-brac/pox virus and zinc finger) domain and codes for 579 amino acids. Limited homology is also detected to the mammalian transcription factor inhibitor IkBa, This implies a close evolutionary relationship between these proteins. To facilitate transformation of crop plants, NPR1 gene was subcloned into a plant transformation vector pHS100 at XbaI and BamHI restriction sites. The recombinant vector pHSAM was mobilized into Agrobacterium tumefaciens LBA4404 by tri-parental mating. A. tumefaciens with recombinant clone pHSAM was used for transforming tobacco. A number of kanamycin resistant plants were generated. Of the 15 plants PCR checked with NPR1 gene specific primers, 5 were positive. These positive plants need to be analyzed further for the expression of NPR1 gene.