Climatic variations have increased the occurrence of heat stress during critical growth stages, which negatively affects grain yield in rice. Plants adapt to harsh environments, and particularly high-temperature stress, by regulating their physiological and biochemical processes, which are key tolerance mechanisms. The 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 indices. Estimates of variance components revealed significant differences (P < 0.001) among genotypes, treatments and their interactions for almost all traits. The principal component analysis showed significant diversity among 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. 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 during early growth. Notably, heat-sensitive mutants accumulated reactive oxygen species, reduced catalase activity and upregulated OsSRFP1 expression under heat stress, suggesting their key roles in regulating heat tolerance in rice. The heat-tolerant mutants identified in this study could be used in breeding programs and to develop mapping populations to unravel the underlying genetic architecture for heat-stress adaptability.