Abstract: To investigate the aroma characteristics and differences among different flue-cured tobacco varieties, this study analyzed the aroma components of nine flue-cured tobacco varieties from the same production region in Yunnan using headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry (HS-SPME-GC/MS), combined with multivariate statistical and odor activity value (OAV) analysis. The results indicated that: (1) A total of 74 volatile aroma compounds were identified across 9 varieties, with variations in compound types and total relative content. The number of aroma compounds was highest in K326 and lowest in Yunyan 301, while the total relative content was highest in K326 and lowest in Yunyan 116. Cluster analysis classified K326, YN223, Yunyan 121, Yunyan 301, and Yunyan 87 into one group, and Yunyan 97, Yunyan 100, Yunyan 105, and Yunyan 116 into another group. (2) Eighteen key aroma compounds were screened by PLS-DA (VIP>1, p<0.05), among which neophytadiene, benzaldehyde dimethyl acetal, benzyl alcohol, and solanone were the core marker compounds (VIP>2). (3) OAV analysis identified 19 aroma-active compounds with OAV>1, including eight common aroma-active compounds (damascenone, damascone, nonanal, β-cyclocitral, megastigmatrienone 1/2/4, and β-ionone) that contributed to the basic fruity, floral, and tobacco-like aroma notes of all varieties. Methyl decanoate, nonanal, geranylacetone, and megastigmatrienone 2 (VIP>1 and OAV>1) were pinpointed as potential characteristic compounds driving the stylistic differences among varieties. This study clarified the differences in aroma characteristics and key aroma-contributing compounds among different flue-cured tobacco varieties grown in the same production area of Yunnan, providing candidate targets for molecular marker-assisted breeding and a theoretical basis for industrial modular formulation design.