The contents of carotenoid and polyphenol in leaves of five flue-cured tobacco cultivars grown in Yunnan in 2009 were quantitatively determined with reverse phase high performance liquid chromatography, and the smoking quality of the tobacco was evaluated. The results showed that: 1) The contents of carotenoid and polyphenol in different flue-cured tobacco cultivars were varied. The carotenoid content in Yunyan85 was the highest, followed by NC297, KRK26 and Yunyan87, while K326 the last. The polyphenol content in Yunyan87 was the highest, followed by NC297, KRK26 the last. 2) The carotenoid contents in middle leaves of Yunyan85, Yunyan87, K326 and NC297 were higher than those in upper leaves, while it was on the contrary for KRK26. The polyphenol contents in upper leaves were higher than those in middle leaves of all cultivars. 3) The smoking quality of Yunyan87, Yunyan85 and KRK26 was superior to that of NC297 and K326. Higher polyphenol content related to lower smoking quality, too high or too low carotenoid content was not good for smoking quality. Preliminary experiments results indicated that within the appropriate range of carotenoid contents, 115-133 μg/g, the lower the polyphenol content was, the better the smoking quality would be.