Global warming-associated increases in temperature, particularly at nighttime, are detrimental to rice yield and quality. Metabolomic profiling was used to examine and compare the short-term extreme high nighttime temperature-induced molecular perturbations in rice ( Oryza sativa) coisogenic strains with contrasting heat-tolerances at the first stage of seed ripening. Compared to the heat-sensitive strain, antioxidant molecules were higher in abundance in the heat-tolerant strain, whereas the abundances of molecules involved in photosynthesis, nucleotide catabolism, and the S-adenosylmethionine (SAM) cycle varied only slightly. Thus, we proposed that the high abundance of antioxidant molecules in the heat-tolerant strain alleviated cellular oxidative stress, which protected photosynthesis, nucleotide catabolism, and the SAM cycle, leading to good grain filling.