Abstract: In this study, 12 counties in 7 provinces of China’s main tobacco cultivation areas were divided into 7 ecotopes based on geographic orientation and latitudinal difference. Then nicotine content variation in flue-cured tobacco under the same cultivation conditions in different ecotopes was analyzed. The results show extensive flue-cured tobacco nicotine content variation for different ecotopes. The degree of inter-annual nicotine content variation is highest for Liaoning and lowest for Henan and Fujian regions. Inter-annual nicotine content stability in the southern regions (Guizhou, Sichuan, Yunnan and Fujian) is higher than in the northern regions (Liaoning, Shandong and Henan). Nicotine content variation in flue-cured tobacco among different stalk parts is highest for Guizhou and Sichuan, but lowest for Liaoning. The characteristics of nicotine content in the stalk parts are more obvious for the southern than the northern regions. Nicotine content variation in the stalk parts is low for Henan region but high for Fujian region. Nicotine content variation is high in middle leaves, low in lower leaves and relatively stable in upper leaves. Nicotine content stabi- lity in the different stalk parts and grades in north are higher than in the south. Nicotine content of orange leaves is higher than lemon-yellow leaves in all the ecotopes. Nicotine content variation among the different regions is mainly driven by variation of nicotine in orange leaves of flue-cured tobacco. Difference in nicotine content from one ecotope to another is significant. Nicotine content of flue-cured tobacco is highest for Henan region and lowest for Liaoning, and is higher for the north than for the south; that in Liaoning region is approaching that in the south, while that in Yunnan region approaching the north.