Climate change poses a major threat to rice productivity, particularly due to high-temperature stress during anthesis, which severely impacts the grain yield. Understanding the physiological and biochemical responses of different rice genotypes to high-temperature stress is critical for breeding resilient varieties. In this study, we assessed two contrasting rice genotypes, high-temperature-tolerant-1 (HTR-1) and high-temperature-sensitive (HTS-5), to confirm previously established physiological and hormonal mechanisms associated with high-temperature tolerance. The study evaluated morphological, physiological, and biochemical markers at the anthesis stage under control (29/24 degrees C) and high-temperature stress (38 degrees C for six hours) conditions. Our results confirmed that HTR-1 exhibits superior tolerance through better antioxidant enzyme activity, higher anther dehiscence, and lower oxidative damage. The genotype HTS-5 exhibited a substantial rise in hydrogen peroxide (1.9-fold) and malondialdehyde (1.74-fold) levels, accompanied by the reduced activity of antioxidant enzymes. Furthermore, the high transcript level of cytosolic APX (OsAPX1, OsAPX2), peroxisomal APX (OsAPX3 and OsAPX4), OsCATA, and OsCATB confirmed high antioxidant activity in HTR-1. Moreover, the GA and IAA levels were reduced in both genotypes, while the ABA concentration was increased significantly in the anthers of HTS-5 as compared to those of HTR-1. This suggests that higher ABA production, along with higher reactive oxygen species (ROS) in the anthers, could lead to sterility in rice under high-temperature scenarios. These findings confirmed HTR-1 as a promising genetic resource for breeding heat-tolerant rice, by validating physiological and biochemical mechanisms of high-temperature resilience. This study also provides practical insights for selecting suitable genotypes to improve rice production under the challenges of climate change.