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High night temperature (HNT) causes substantial yield loss in rice (Oryza sativa L.). In this study, the physiological processes related to flag leaf dark respiration (Rn) and grain filling under HNT were explored in a multi‐parent advanced generation intercross population developed for heat tolerance (MAGIC^heat) along with selected high temperature tolerant breeding lines developed with heat‐tolerant parents. Within a subset of lines, flag leaf Rn under HNT treatment was related to lower spikelet number per panicle and thus reduced yield. HNT enhanced the nighttime reduction of non‐structural carbohydrates (NSC) in stem tissue, but not in leaves, and stem nighttime NSC reduction was negatively correlated with yield. Between heading and harvest, the major difference in NSC concentration was found for starch, but not for soluble sugar. HNT weakened the relationship between NSC remobilization and harvest …