By using gas chromatography, the authors measured and analyzed the contents of four major alkaloids, including nicotine, nornicotine, anabasine, anatabine in flue-cured tobacco from Hunan, Guizhou, Yunnan and Shandong Province. Results showed that the relative standard deviations were below 3.5% and the average recovery of alkaloids were 98.6% - 103.0%. The content of alkaloids from high to low was nicotine＞nornicotine＞anatabine＞anabasine, and the average level of total alkaloid in four production regions was in the sequence: Hunan＞Guizhou＞Yunnan＞Shandong, which was also closely related with cultivars. Based on the statistical analysis of data, leaves from the four major production regions were significantly different in major alkaloids and total alkaloid at 0.01 level (except for nornictine), which also held true in the proportions of anabasine and anatabine in total alkaloid.