In order to investigate characterization and the differences of volatile substances in different types of flue-cured tobacco, the volatile components (VOCs) and relative contents of six varieties of flue-cured tobacco were analyzed by headspace gas chromatography-ion mobility spectrometry (HS-GC-IMS) and headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). Combined with the relative odor activity value (ROAV), important flavor substances were determined and different aroma components were screened by multivariate statistical analysis. The results showed that 93 and 80 kinds of compounds, such as esters, alcohols, and aldehydes, were detected by GC-IMS and HS-SPME-GC-MS in six flue-cured tobacco varieties, respectively. A total of 19 volatile flavor compounds, such as benzaldehyde, acetic acid, valeraldehyde, and methyl acetate, were selected as compounds to distinguish the aroma of different toasted tobaccos by orthogonal partial least-squares discrimination analysis (OPLS-DA). In this study, more comprehensive information about volatile flavor components of six flavor types of flue-cured tobacco has been obtained by the synergistic analysis of two distinct analytical instruments. Meanwhile, this study clarified the composition and differences of aroma components of six types of flue-cured tobacco, which lays a foundation for the flavor control of different types of flue-cured tobacco.