The contents of free and combined campesterol, stigmasterol and ß-sitosterol in tobacco leaves of different types, varieties, growing areas and stalk positions were determined by gas chromatography via hydrolysis by concentrated hydrochloric acid, saponification by an alcoholic KOH solution, extraction with hexane, and derivation with bis(trimethylsilyl)trifluoroacetamide (BSTFA). The results showed that the limits of detection (LODs) of the sterols varied from 0.10 to 0.35 µg/mL and their recoveries ranged from 87% to 99% with the relative standard deviations (RSDs) of 2.3% to 3.3%. The total phytosterol content in tobacco leaf was greatly affected by tobacco type, stalk position and growing area. The distribution of different kinds of phytosterol in tobacco leaf was also influenced by tobacco variety, while the effects of stalk position and growing area were not obvious. The contents of steryl ester (SE), steryl glycoside and acylated steryl glycoside (SG+ASG) might be positively correlated with the contents of higher fatty acids and monosaccharide in tobacco leaf, respectively.