In groundnut, glyphosate resistant plants (cul. TMV-2) were developed by over expressing pEGADEPSPS with altered kinetics of enzyme, which do not bind to glyphosate. Agrobacterium mediated in planta transformation adopted to develop transgenic groundnut lines expressing EPSPS. Single leaflet glyphosate induced chlorosis bioassay was standardized and used to assess the glyphosate resistance in groundnut transgenic lines of T1 and T2 generations. The T1 generation plants grown under transgenic housing facilities along with wild type and their relative tolerance analyzed by the leaf swabbing technique indicated the integration of the transgene in tolerant plants by PCR. The T2 generation plants screened for glyphosate resistance by swabbing 3000 ppm of glyphosate at 45 DAS observed that 30% of transgenic plants showed some degree of yellowing and leaf mortality and resistance confirmed by PCR. The chlorophyll degradation was less in transgenic and also maintained higher membrane integrity compared to wild type plant