Abstract: To elucidate the causes of industrial usability differences in upper tobacco leaves, this study employed untargeted metabolomics to analyze upper flue-cured tobacco samples of varying industrial usability from three aroma types: robust, medium, and fresh. We identified differential biomarkers, their enriched pathways, and analyzed their relationship with sensory quality. The results showed that all upper tobacco leaf samples used in the experiment were classified as Class I or Class II industrial usability, with no Class III samples. Sugars, lipids, organic acids, amino acids, and alkaloids constituted a high proportion of compounds in the three aroma types of tobacco leaves. Based on the criteria of VIP ≥ 1.0 and FC < 0.5 or FC > 2.0, five and seven differential biomarkers were screened for robust and fresh aroma types with different industrial usability in 2022, respectively, while ten and four differential biomarkers were identified for 2023, respectively. These biomarkers belonged to lipids, amino acids, alkaloids, organic acids, sugars, and phenolic compounds. In the robust aroma producing region's upper tobacco leaves from both 2022 and 2023, L-glutamine was a common differential biomarker, whereas no common differential biomarkers were found in the fresh aroma type. Further analysis of the correlation between differential biomarkers and sensory quality revealed that the accumulation of amino acids, lipids, and phenolic compounds positively regulated the industrial usability of upper tobacco leaves in the robust aroma producing region, while the accumulation of lipids and amino acids negatively regulated the industrial usability of upper tobacco leaves in the fresh aroma producing region. Differential metabolite enrichment pathway analysis indicated that the 26 differential biomarkers identified in this study were significantly and primarily enriched in amino acid metabolism-related pathways. L-glutamine is likely a core differential biomarker influencing the industrial usability of robust aroma tobacco leaves.