Abstract: To investigate the metabolic characteristics of tobacco leaves from high-altitude areas in Bijie City, combined metabolome and transcriptome analysis was performed on the leaves of cv. Jiucaiping No. 2 and Yunyan 87 from three tobacco growing areas in Bijie with an altitude above 1 800 m. The differential metabolites and genes between Jiucaiping No. 2 and Yunyan 87 were identified by comparative analysis, and the key gene modules contributing to the differences were identified by weighted gene co-expression network analysis. The results showed that there were metabolic differences between Jiucaiping No. 2 and Yunyan 87 tobacco leaves from the three tobacco growing areas, and the flavonoid metabolite isorhamnetin was highly accumulated in Jiucaiping No. 2 leaves. A total of 265 common differentially expressed genes were identified in the leaves, which were mainly enriched in the flavonoid biosynthetic pathway and the flavone and flavonol biosynthetic pathways. Nine gene modules related to isorhamnetin were identified by the weighted gene co-expression network, two of which were significantly enriched in the flavonoid biosynthetic pathway and the flavone and flavonol biosynthetic pathways. Further integrated analysis revealed that the down-regulated expression of NtPAL1, NtCHIL, NtF3H, NtTT7 and NtFLS1 genes in Jiucaiping No. 2 leaves might lead to the low accumulation of phenylalanine, kaempferol and its derivatives, quercetin and its derivatives in tobacco leaves, while the up-regulated expression of methyltransferase genes such as NtASMTs, NtROMTs and NtCCoAMT might promote the high accumulation of isorhamnetin in tobacco leaves. This study tentatively demonstrates that the differential changes in flavonoid-related metabolic pathways have a certain impact on the high-altitude adaptation of Jiucaiping No. 2, and the results provide theoretical guidance for the in-depth analysis of the growth adaptation mechanism of Jiucaiping No. 2 in high-altitude areas.