A large number of non-volatile metabolites are produced during the growth of rice, but there are few studies on the changes of metabolites at different harvest times. In this study, Nangeng 9308 used taken as the research object. Liquid chromatography-mass spectrometry (LC-MS) was used to analyze japonica rice grown in Nanjing, Huai'an, and Lianyungang by non-targeted metabolomics. This study focused on the effects of different harvest times (50, 55 and 60 days after heading) on the metabolite composition of rice. The results showed that a total of 2 111 metabolites were identified. The major metabolic pathways were autophagy-other, ABC transporter, and glycerophospholipid metabolism. In addition, harvest time has a significant effect on the metabolite composition of rice, especially the content of amino acids and sugars. The content of amino acid and sugar in late-harvested rice were generally higher than those in early-harvested rice. In addition, L-glutamic acid was in an upward trend in japonica rice grown in the three regions. This study provides a reference for optimizing rice harvest time and improving rice quality by analyzing non-volatile metabolite changes.