Introduction: Tobacco is the most widely grown non-food crop in the world. Nicotine is the most abundant volatile alkaloid in tobacco leaves. Objectives: This work aimed at measuring nicotine levels in the leaves of seven different varieties of Nicotiana tabacum, namely: Virginia, Burlip, Katrina, Shk al-bent, Zegrin, Basma and Burley, cultivated in Syria. Methods: Nicotine was extracted according to approved method and its concentration was determined by LC/MS/MS in comparison with a standard material dilution series. The percentage of nicotine concentration was calculated manually. Statistical analysis was used to assess the significance of differences among variables and to perform multiple comparisons. Results: The amount of nicotine in dry weight of tobacco leaves represented 6.7% in Virginia variety, 4.9% in Burlip, 4.84% in Katrina, 4.67% in Shk al-bent, 4% in Zegrin, 3.3% in Basma and <3% in Burley. Significant differences in nicotine concentration were found among the different varieties as determined by LSD test at a level of 0.05. Conclusion: This study shows the importance of tobacco varieties grown in Syria, particularly Virginia, Burlip, Katrina, as a cheap and wealthy source for nicotine to be used in some industries.