The oxidative stress response was studied in five different clones of Saccharum spontaneum (IMP-564, IS76-216, Pamba, SES-90 and Taiwan) by analyzing the physiological and biochemical parameters and gene expression pattern. The sixty days-old plants were sprayed with different concentrations of 30% H2O2 (300 ppm, 500 ppm and 1000 ppm) for consecutive three days in glass house. Adaptive response was recorded in terms of chlorophyll fluorescence, chlorophyll stability index (CSI), activities of peroxidase (POX) and super oxide (SOD), lipid peroxidation (LPO), protein and proline content at 48 h and 72 h of H2O2 treatment. The IMP -564 clone recorded high SOD and lower lipid peroxidation levels. Based on chlorophyll fluorescence, CSI, peroxidase, SOD and lipid peroxidation values, and the clone SES-90 recorded positive trend for stress tolerance. CSI showed significant difference between genotypes and different concentrations of H2O2. As the genotypes IMP-564 and SES-90 showed positive response to oxidative stress tolerance, the S. spontaneum genotype SES-90 was selected for comparative gene expression studies with Erianthus sp clones IJ76-389 and IK76-91. Different concentrations of H2O2 and different time intervals (control, 500 ppm and 1000 ppm for 48 h and 72 h) were used for gene expression studies. The stress responsive genes viz. Ascorbate peroxidase gene (Apx) Nam, Ataf1 and Cuc2 gene (NAC), Ethylene response factor gene (Erf), Glutathione S-transferases gene (Gst), Myeloblastosis antisense gene (Mybas) and Catalase gene (Cat) showed differential expression among the genotypes of S. spontaneum and Erianthus sp. Among the genotypes, SES 90 and IK76-91 showed better tolerance against oxidative stress and the expression levels of stress responsive genes in the genotype IK76-91 was higher than the genotype SES 90.