Abstract: To study the variability of chemical components in flue-cured tobacco stems, stems of different forms after threshing and redrying of major tobacco cultivars in Qujing tobacco planting areas were gathered and analyzed by a continuous flow analyzer, high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) methods. The results showed that: 1) There were differences in routine chemical components, cell wall substances and aroma components in the tobacco stems of different cultivars, however no obvious trend was found. 2) The mass fractions of lutein, β-carotene, rutin, aroma component precursors and the total amount of aroma components in fragmentary stems were significantly higher (P < 0.05) than those in stem head, long and short stems. The mass fractions of total alkaloids, chlorogenic acid, scopoletin and petroleum ether extract in stem head were significantly higher than those in long, short and fragmentary stems (P < 0.05). 3) Cluster analysis results indicated that according to stem form, tobacco stems could be mainly classified into three categories: stem head, long and short stems, and fragmentary stem. Long and short stems had higher mass fractions of sugar and potassium and lower alkali and lignin with more harmonized chemical components. It seemed that priority to the use of long and short stems as the material of cigarette blend would be conducive to improve the compatibility and use value of stems with materials in cigarette blends. The stem heads and fragmentary stems with higher mass fractions of alkali and aroma components could be used as preferred materials for e-cigarette nicotine extraction and reconstituted tobacco preparation.