In order to compare the secondary metabolite contents in flue-cured leaves from different areas in Guizhou Province, the leaf samples of C3F and B2F grades of K326, Yunyan 85, Yunyan 87, Guiyan 1 and Guiyan 201 varieties were collected from Daozhen, Weining, Panxian, Tianzhu and Guiding counties, and the secondary metabolite contents in the samples were analyzed. The results showed that: 1) The average contents of main organic acids, polyphenols, alkaloids and sterols were 112.47, 33.65, 29.79 and 2.47 mg/g, respectively. 2) The contents of organic acids, polyphenols and alkaloids in flue-cured tobacco leaves from Guizhou were relatively higher, while the contents of sterols were moderate comparing with those from other growing areas in China. 3) The total content of secondary metabolites in leaves from Panxian was the highest, followed by Weining, Tianzhu and Daozhen, while that from Guiding was the lowest. The highest organic acid content was found in leaves from Panxian, while the highest polyphenol content in leaves from Weining and the highest alkaloid content in leaves from Tianzhu.