Abstract: A liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for simultaneous quantification of nine water-soluble B vitamins in tobacco was established by adopting multi-reaction detection mode (MRM). The parameters, such as pH value of mobile phase, column temperature, extraction solvent and volume, were optimized. Tobacco leaf samples collected from three growing areas were analyzed with the developed method. The results showed that:1) There was a good linear relationship for the nine B vitamins with the determination coefficient (R²) of 0.994 6-0.999 9. 2) The recoveries of the nine B vitamins ranged from 91.00% to 108.79%. The intra-and inter-day relative standard deviations (RSDs) were 1.25%-9.73%. The limits of quantification (LOQ) for all the analytes were from 0.01 to 0.50 ng/mL. 3) The contents of B vitamins differed among the leaves from the three growing areas. The contents of VB₂, VB₇ and VB₉ in tobacco leaves from Xiangxian County in Henan were higher than those of the others, and the VB₃ content in tobacco leaves from Guiyang City in Guizhou was higher. This method is simple in operation and features good repeatability and high sensitivity, it can be used for the simultaneous analysis of the nine B vitamins.