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Further dissection of physiological molecular mechanisms is indispensable to alleviate rice yield losses resulting from cold injury. By using 105 near-isogenic lines (NILs) derived from a backcross between cv. Lijiangxintuanheigu (LTH) and cv. Towada, we detected quantitative trait loci (QTLs) for physiological traits of the rice flag leaf, based on polymorphic simple sequence repeat (SSR) markers, inclusive composite interval mapping (ICIM), mixed composite interval mapping (MCIM) approaches and phenotypic value subjected to combine with cold-water stress and three nitrogen application rates. By using ICIM, a total of 34 QTLs with additive effects (A-QTLs) were identified on chromosomes 1, 3, 4, 5, 6, 7 and 10, and the phenotypic variation (R2) explained by each QTL ranged from 8.46 to 29.14%. By using MCIM, 20 A-QTLs and 14 pairs of QTLs with epistatic × environment interaction effects (Epistatic QTLs) were detected, the contribution of environment interaction (H2AE) was 0.87 to 7.36%, while the contribution rates of E-QTL were from 0.97 to 3.58%. Fourteen A-QTLs were detected by ICIM and MCIM, which may serve as a basis for fine-mapping and candidate gene studies, and providing strategies for the development of cold-tolerant rice cultivars and nitrogen application to alleviate chilling stress.