Abstract: In order to study the correlation between contents of chemical substances and altitude, 182 of flue-cured tobacco cutter leaf samples were collected from different altitudes in Yunnan. The sampling altitude ranged from 706 m to 2356 m and the tobacco cultivars included Yunyan87, Yunyan85, K326 and Honghuadajinyuan (HD). The main chemical substances and aroma precursors such as organic acids, polyphenols, were detected in these samples. The difference in chemical substance contents and their correlation with altitude were studied through correlation and cluster analysis. The results showed that the contents of soluble sugars, organic acid in HD were the lowest in four tobacco cultivars, while total nitrogen, nicotine, plastid pigments and polyphenols were the highest. The correlations between contents of total sugar, total nitrogen, nicotine and petroleum ether extracts and altitude in K326 were different from the other three tobacco cultivars. The same results were found in the correlation between organic acids (malonic acid, succinic acid, linoleic acid), plastid pigments (beta renieratene), monosaccharide and disaccharide and altitude, respectively. Therefore, the change of chemical substances in flue-cured tobacco leaves with tobacco planting area altitude is greatly affected by genotypes.