Abstract: The non-volatile organic acid components affected the sensory features of flue-cured tobacco significantly. To study the differences in non-volatile organic acid content in flue-cured tobacco from different growing areas and the relationship with sensory features, non-volatile organic acids in flue-cured tobacco samples from five different growing areas (Heilongjiang, Sichuan, Hunan, Hubei and Yunnan) were detected by ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). The differences in non-volatile organic acids from different growing areas were obtained and the metabolic pathways of characteristic difference components were determined by using orthogonal partial least-squares discrimination analysis (OPLS-DA) model, VIP values, ANOVA and KEGG enrichment pathway analysis. As shown by the results, a total of 69 non-volatile organic acids were detected, including 23 polybasic acids, 21 free fatty acids, and 25 other organic acids. The OPLS-DA model based on non-volatile organic acids had an ideal effect in distinguishing tobacco from different growing areas, and a good fitting result was obtained with the parameters R²Y of 0.931 and Q² of 0.595 respectively. 26 characteristic difference non-volatile organic acids were screened out through the model's VIP values and ANOVA, and 16 metabolic pathways associated with the above 26 components were obtained using KEGG enrichment analysis. The characteristic difference non-volatile organic acid content of tobacco samples from Mudanjiang growing area in Heilongjiang was the most different from others, followed by Hengyang growing area in Hunan. The characteristic difference non-volatile organic acids in tobacco from growing areas of Liangshan in Sichuan, Chuxiong in Yunnan, and Enshi in Hubei had little difference between groups, which were consistent with the sensory features of flue-curred tobacco from the tobacco growing areas.