Thirteen tobacco materials (K326, VA116, G70, NC82, GDH94, GDH88, Jiucaiping2, TN90, Nanjiang3, Qinggeng, Basma, Bina1, Meitandaman tobacco) were used as parents, and 40 cross combinations were designed by NCII incomplete diallel cross design. The stem percentage of parents and F1 hybrids was measured, and the heritability, combining ability and heterosis performance of stem percentage of tobacco leaves were analyzed. The results showed that there were significant or extremely significant differences in stem percentage among the hybrid combinations; the broad heritability and narrow heritability of stem percentage were 96.11% and 72.32% respectively, which indicated that the trait transmissibility was strong and the reliability of genotype selection by phenotype was high; the variance of general combining ability and special combining ability were both extremely significant, which indicated that the stem percentage was controlled by both additive and non-additive effects. The variance of general combining ability was greater than that of special combining ability; and the negative effect value of general combining ability of the three parents GDH88, Qinggeng and Meitandaman tobacco was small, which was good parents for improving tobacco stem percentage. The heterosis of leaf stem percentage was mainly negative mid-parent heterosis and negative super-parent heterosis, accounting for 77.5% of all hybrid combinations. Hybrid combinations VA116 × GDH88, K326 × GDH88 and VA116 × Jiucaiping2 showed better special combining ability and heterosis, which are good for selecting varieties with low stem percentage.