Abstract: To clarify the differences in flavonoid metabolites and metabolic pathways of tobacco leaves from four major tobacco growing areas in Sichuan Province, the metabolomics of flavonoid metabolites in mature tobacco leaves from these regions were studied by LC-MS. The different effects on tobacco metabolites were investigated by combining principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) with metabolic pathway analysis. The results showed that: 1) A total of 290 flavonoid metabolites were detected, which were divided into 9 categories, including flavonols, flavonoids, dihydroflavonoids, anthocyanins, dihydroflavonols, chalcones, isoflavones, flavanols, and dihydroisoflavones. 2) There were 18 different metabolites between Liangshan and Panzhihua leaves, 30 between Liangshan and Yibin leaves, 38 between Liangshan and Guangyuan leaves, 19 between Panzhihua and Yibin leaves, 45 between Panzhihua and Guangyuan leaves, 23 between Yibin and Guangyuan leaves. 3) The OPLS-DA results showed that tobacco leaves from the different areas could be clearly distinguished and there were significant sample accumulation by areas, indicating that the growing area greatly influenced the flavonoid metabolism in tobacco leaves. 4) The metabolic pathway analysis showed that the differential metabolites of tobacco leaves from these different areas in Sichuan were mainly annotated and enriched in six major metabolic pathways, including metabolic pathways, flavonoid and flavonol biosynthesis pathways, secondary metabolite biosynthesis pathways, isoflavone biosynthesis pathways, flavonoid biosynthesis pathways, and anthocyanin biosynthesis pathways. The flavonoid metabolites in tobacco leaves from the different growing areas were different, giving these tobacco leaves different flavor notes.