The levels of sugars in tobacco are important for leaf characterization. They contribute substantially to the sensory properties of cigarettes, and generate by pyrolysis undesirable compounds such as aldehydes. More modern analyses of sugars have been performed using HPLC, but their molecules do not have chromophors and detection is typically performed using a refractive index detector, which is not very sensitive and allows identification based only on retention time. The high hydrophilic character of sugar molecules requires the use of special columns such as Rezex™ or HILIC-amino for separation. A new procedure for the quantitation of sugars has been developed using a new HILIC type column and a tandem mass spectrometer for detection. The procedure allows the separation and analysis of several C5 and C6 monosaccharides, several disaccharides, sorbitol, as well as several inositols. The procedure uses a YMC-Pack Polyamine II column, and the separation is performed in isocratic conditions using mobile phase with 67% CH3CN and 35% H2O, that has 80 M Cs(CH3COO). The mass spectrometer selects in Q1 the adduct molecule of Cs+ with the sugar, and in Q3 detects the Cs+ ions. This procedure has been used for the quantitation of fructose, glucose, sucrose and myo-inositol in 15 different tobaccos. Although based on a limited number of tobacco types, the study provides a comparison of flue-cured, burley, and Oriental tobaccos, leaf + some tips vs. lower stalk, and different growing locations. The results show the expected differences between flue-cured, burley, and Oriental(s). Also, it showed that the growing location for the tobacco alters the ratios of the levels of different sugars, but maintains rather similar profiles for the same tobacco type.