Plants adapt to harsh environments particularly high temperature stress by regulating their physiological and biochemical processes, which are key tolerance mechanisms. Thus, identification of heat-tolerant rice genotypes and reliable selection indices are crucial for rice improvement programs. Here, we evaluated the response of a rice mutant population for high-temperature stress at the seedling and reproductive stages based on agronomic, physiological and molecular traits. The estimate of variance components revealed significant differences (P<0.001) among genotypes, treatments and their interaction for almost all traits. Principal component analysis showed significant diversity among the genotypes and traits under high-temperature stress. The mutant ‘HTT-121’ was identified as the most heat tolerant mutant with higher grain yield, panicle fertility, cell membrane thermo-stability (CMTS) and antioxidant enzyme levels under heat stress conditions. Various seedling-based morpho-physiological traits (leaf fresh weight, relative water contents, malondialdehyde, CMTS) and biochemical traits (superoxide dismutase, catalase and hydrogen peroxide) explained variations in grain yield that could be used as selection indices for heat tolerance in rice at early growth stages. Notably, heat sensitive mutants showed a significant accumulation of ROS level, reduced activities of catalase and upregulation of OsSRFP1 expression under heat stress, suggesting their key role in regulating heat tolerance in rice. The heat-tolerant mutants identified in this study could be used in breeding programs and the development of mapping populations to unravel the underlying genetic architecture for heat-stress adaptability.Summary text for table of contentsHeat stress probably due to changing climate scenario has become a serious threat for global rice production. On the other side, efforts to develop high yielding cultivars have led to the reduced genetic variability to withstand harsh environmental conditions. This study aimed to identify novel heat tolerant mutants developed through gamma irradiation which will provide a unique genetic resource for breeding programs. Further, we have identified reliable selection indices for screening heat-tolerant rice germplasm at early growth stages.