Abstract: In order to investigate the variation of crotonaldehyde release characteristics in mainstream smoke of Henan single grade tobacco, 144 flue-cured tobacco samples of 3 stalk positions (upper, middle and lower) were collected from the main tobacco-growing areas in Henan Province. Ccrontonaldehyde content in mainstream smoke was determined with high performance liquid chromatography and was studied by cluster analysis. The results showed that the average crontonaldehyde content in mainstream smoke in upper, middle and lower leaves were 21.86, 23.72, 23.91 μg/cig, respectively, with the middle leaves showing the largest variation. The crontonaldehyde contents in upper and lower leaves showed significant difference, while no significant difference was observed between middle leaves and lower, upper leaves. For the leaves from the same stalk position, main tobacco cultivars in the order of crontonaldehyde content in mainstream smoke were Qinyan96 > Zhongyan203 > Yuyan10 > Zhongyan100. The crontonaldehyde contents of lower and middle leaves differed significantly between Qinyan96 and Zhongyan100, but this difference was not significant between the other varieties or between upper leaves of all the tested varieties. The results of cluster analysis suggested that crontonaldehyde contents in mainstream smoke of flue-cured tobacco leaves from Henan Province could be divided into three categories (high, middle and low), with the high release type mainly distributed in Sanmenxia, Luoyang and Nanyang most of the producing areas, the middle release type mainly distributed in Luohe, Pingdingshan, and Xuchang most of the producing areas, the low release type only distributed in Pingdingshan and small part of Luohe producing areas. This result can provide a theoretical basis for the harm reduction of cigarettes.