Abstract: In order to study the organic acids in different tobacco leaves, a GC-MS/MS method of high throughput and high sensitivity was established. The results showed that:1) This method could simultaneously determine 42 organic acids in tobacco and accurately quantify 8 of them (such as isobutyric acid, 4-oxononanoic acid, 3-phenylpropionic acid, etc.) that could not be quantified by GC-MS. 2) The intra-day and inter-day precisions for all the analytes were less than 12%, the average recoveries ranged from 76% to 110%, and the limits of detection (LODs) of most acids were less than 10 ng/mL. 3) For 3 flue-cured tobacco leaves of different flavor types (fresh, medium, robust), the contents of 21 acids differed significantly. The contents of pentanoic acid, hexanoic acid, benzoic acid, phenylacetic acid and so on were the highest in flue-cured tobacco of the robust flavor type, the contents of isovaleric acid, lactic acid, 3-hydroxypropionic acid and so on were the highest in flue-cured tobacco of the medium flavor type, and the contents of 4-oxononanoic acid and 2-methoxybenzoic acid were the highest in flue-cured tobacco of the fresh flavor type. 4) In flue-cured, burley, oriental, sun-cured tobacco samples, the contents of 34 acids differed significantly. Most of the acid components were the highest in oriental tobacco and the lowest in flue-cured tobacco, and their contents in burley and sun-cured tobacco were moderate except that benzoic acid, isovaleric acid and crotonic acid were the highest in burley tobacco, while 2-octenoic acid and 2-methoxybenzoic acid were the highest in sun-cured tobacco. This method features high sensitivity, precision and recovery, and could be used in the simultaneous analysis of organic acids in tobacco leaves of different flavor types or different type tobacco.