The most effective way to produce a plant resistant to glyphosate is manipulation of EPSPS enzyme in order to reduce its affinity to this herbicide. It is one of the vital enzymes in the shikimate pathway, which is responsible for producing aromatic amino acids and various secondary metabolites in plants and bacteria. Without an active enzyme, the organism will not be able to survive. In the present study, one of the important domains in the EPSPS enzyme was modified by site-directed mutagenesis using mega-primer method and one tube approach. Glycine in position 96 was modified to Alanine which was sub-cloned along with the wild-type gene into pPZPY122 plant binary vector. Hashemi cultivar of rice (Oryza sativa) was used for transformation using EHA105 strain of Agrobacterium tumefaciens by in planta method. T₂ transgenic plants separately were evaluated to resistance to the gentamicin and glyphosate. Also, positive gene expression was confirmed by Quantitative PCR. Therefore, based on the results, these plants showed significantly a high degree of resistance to the herbicide.