<p>Rice is important staple crop of world’s most densely populated regions, affected by increase in flood conditions due to climatic change. It causes reduction in rice yield due to submergence stress. Identification and development of submergence tolerance high yielding rice varieties can counter this challenge. Thereby, the present study was conducted to evaluate a substantial collection of rice germplasm to identify submergence-tolerant rice lines using biochemical and molecular assessments. A total of 116 rice lines, along with tolerant (FR13A) and sensitive checks (DRR44), were grown in an augmented block design with two replications and screened under submerged conditions. Twenty-five lines exhibited submergence stress tolerance based on morphological characteristics and survivability scores. Furthermore, these rice lines, along with contrasting checks, were screened for submergence tolerance based on biochemical and molecular validation. In the submerged scenario, all rice lines showed a decrease in total chlorophyll content compared to the controlled settings; however, the decrease was less prominent in lines such as R17A2977, IR18A1383 and the tolerant check. Further, these rice lines along with checks showed reduction in protein content ranging from 26.3% (IR18A1558) to 52.94% (IR17A2906), which is crucial for survival under submerged conditions. Reduction in soluble and insoluble carbohydrate concentrations was also observed in these lines which ranged within 57.73–84.02% and 50.73–90.32%, respectively, depicting reduced photosynthesis and increased starch mobilization for energy availability to survive submergence stress. Reduction in amylase enzyme content ranged within 10–51% under submergence stress. Significant increase in superoxide dismutase (SOD) levels was found in all the rice lines within the range of 54–67%. Molecular validation using eight <i>Sub1</i> (submergence) gene specific SSRs exhibited two SSRs i.e. RM316 and RM8300 that differentiated tolerant and sensitive checks in this study. Overall, lines such as IR15F1896, IR18A1876, IR18A1967 exhibited submergence tolerance through both the marker as well as biochemical analysis that could be used for generating breeding materials for future research aimed at producing submergence stress resilient rice varieties.</p> Graphical abstract <p>Workflow for screening submergence tolerance in rice. From an initial 116 lines, 25 tolerant lines were identified following extended submergence. These selected lines were subjected to rigorous physiological and biochemical profiling, alongside molecular validation of the SUB1 locus. It enabled the identification of elite breeding donors for the development of climate-resilient rice varieties. This approach integrates comprehensive biochemical assessment with genetic confirmation to advance breeding strategies aimed at improving submergence tolerance and climate adaptability in rice.</p> <p></p>