Abstract: In order to investigate the distribution of free sterols in tobacco leaf samples of different types, varieties and stalk positions from different planting areas in China, the contents of five sterols (stigmasterol, β-sitosterol, campesterol, cholesterol and ergosterol) in 47 tobacco samples, including flue-cured, cigar and sun-cured tobacco were determined by an ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) method. The results showed that:1) The total contents of free sterols in the samples presented certain regularity, stigmasterol content was the highest in the tobacco samples, followed by β-sitosterol, campesterol, cholesterol and ergosterol. 2) The contents of β-sitosterol in flue-cured and sun-cured tobacco were higher than those of campesterol, while the content of campesterol was higher than that of β-sitosterol in cigar tobacco. 3) The contents of free sterols in tobacco leaves differed among varieties, stalk positions and planting areas.