Abstract: In order to clarify the differences in flavonoids metabolites and metabolic pathways in tobacco leaves from four major tobacco producing areas in Sichuan, LC-MS was used to study the flavonoids metabolites in mature tobacco leaves from four major tobacco producing areas in Sichuan.Combined with principal component analysis ( PCA ) and orthogonal partial least squares discriminant analysis ( OPLS-DA ) and other multivariate statistical analysis and metabolic pathway analysis, the effects of producing areas on tobacco metabolites were discussed. The results showed that : 1 A total of 290 flavonoid metabolites were detected, which were divided into nine categories : flavonols, flavonoids, dihydroflavones, anthocyanins, dihydroflavonols, chalcones, isoflavones, flavanols and dihydroisoflavones. There were 18 differential metabolites between Liangshan and Panzhihua tobacco leaves, 30 differential metabolites between Liangshan and Yibin tobacco leaves, 38 differential metabolites between Liangshan and Guangyuan tobacco leaves, 19 differential metabolites between Panzhihua and Yibin tobacco leaves, 45 differential metabolites between Panzhihua and Guangyuan tobacco leaves, and 23 differential metabolites between Yibin and Guangyuan tobacco leaves. The results of OPLS-DA showed that the tobacco leaves from different producing areas were obviously distinguished, and there were significant sample clusters, indicating that the producing area had a great influence on the flavonoid metabolism of tobacco leaves. The results of metabolic pathway showed that the differential metabolites of tobacco leaves from different producing areas in Sichuan were mainly annotated and enriched in six major metabolic pathways, including metabolic pathway, flavonoid and flavonol biosynthesis pathway, secondary metabolite biosynthesis pathway, isoflavone biosynthesis pathway, flavonoid biosynthesis pathway and anthocyanin biosynthesis pathway. The different origin of tobacco leaves, flavonoids metabolites are also different, but also gives the tobacco different flavor.