Abstract: C3F flue-cured tobaccos come from different production regions in Yunnan and Hunan were used for this study, the content of volatile and semi-volatile acid in flue-cured tobacco were studied by simultaneous distillation and extraction and gas chromatography. The results indicated that: (1) the content of volatile acid in Yunnan and Hunan exist greatly difference, but the content of semi-volatile acid differed little between the Yunnan and Hunan. Most content of volatile acid in Yunnan were all richer than those in Hunan, but the content of semi-volatile acid contrarily. (2) Most content of volatile acid in Qujing and Wenshan were significantly higher than those in Kunming and Zhaotong. The content of semi-volatile acid (except tetradecanoic acid) of Qujing were the highest, followed by Wenshan, while the Kunming were the lowest, and the difference was obviously. (3) Most content of volatile acid (except butyric acid) were significantly different among the different production regions in Hunan, but the difference had no obvious regularity. Most content of semi-volatile acid (except tetradecanoic acid) of Cili were the highest, while the Shaoyang were the lowest, and the difference was obviously.