High-yielding Indian cotton varieties are not amenable for regeneration and transformation because they are recalcitrant in nature. In this work, we have developed Narasimha (NA1325) cotton variety by introducing three Cry genes driven by three different promoters conferring insect resistance. The meristematic region of embryo axis explants were infected and co-cultivated with Agrobacterium tumefacience (LBA4404) harbouring pMDC100 vector with three Cry gene cassettes (alpha-globulin : Cry2Ab, DECaMV35s : Cry1F and nodulin : Cry1Ac) with Npt II as a selectable marker gene. Out of 1010 embryo axes explants infected, 121 (T0) regenerated under two rounds of kanamycin selectionmedium.About 2551T1 seedswere collected from111T0 plants and these seeds screened again with kanamycin at seedling stage. The transgenic plants were characterized by PCR, real time quantitative PCR, lateral flow strip protein assay and insect bioassay. Out of 145 kanamycin resistant plants (T1), twelve showed amplification of all four transgenes: Npt II, Cry2Ab, Cry1F and Cry1Ac through PCR with expected amplicons as 395, 870, 840 and 618 bp, respectively. Further, lateral flow strip test revealed Cry1F and Cry1Ac proteins accumulated in 12 plants, whereas Cry2Ab protein was detected in eight only. The transcripts of all three Cry genes were accumulated significantly higher in transgenic plants at T2 generation. The transgenic lines showed effective resistance againstHelicoverpa armigera and Spodoptera litura larvae. The T2 line L-3 exhibited highest percentage of insect mortality, in which transcripts of all cry genes were accumulated higher than other plants. The transgenic cotton plants carrying triple Cry genes could be an excellent germplasmresource for the breeders for introgressions.