Abstract: This study investigated the effects of exogenous methyl jasmonate (MeJA) on nicotine synthesis and distribution in different parts of tobacco seedlings. Using flue-cured tobacco varieties Zhongchuan208 and Zhongyan100 as test materials, a two-factor (variety × concentration) completely randomized design was adopted, with three MeJA concentrations: 0.1 mg/L (M1), 1 mg/L (M2), 10 mg/L (M3), and a control (CK, sprayed with water mixed with solvent). The results showed that MeJA application significantly increased nicotine content and accumulation in all parts of the seedlings, with the most pronounced enhancement observed at 1 mg/L (M2). Compared to CK, Zhongchuan208 exhibited increases of 0.17, 0.19, 0.35, 0.38, and 0.51 percentage points in nicotine content in roots, stems, lower leaves, middle leaves, and upper leaves, respectively, while Zhongyan100 showed increases of 0.13, 0.21, 0.39, 0.61, and 0.71 percentage points in the corresponding parts. Nicotine accumulation in Zhongchuan208 increased by 57%, 26%, 59%, 52%, and 66% in roots, stems, lower leaves, middle leaves, and upper leaves, respectively, whereas Zhongyan100 displayed increases of 41%, 32%, 50%, 75%, and 61%. MeJA upregulated the expression of key nicotine synthesis enzyme genes, NtPMT and NtQPT. It also significantly enhanced the nicotine synthesis rates per unit root length and root mass. Under the 1 mg/L (M2) treatment, these rates increased by 60% and 66% in Zhongchuan208, and by 76% and 78% in Zhongyan100, respectively. However, it had no significant effect on nicotine distribution ratios among different parts. In conclusion, 1 mg/L MeJA is the optimal concentration for promoting nicotine synthesis, as it significantly increases nicotine accumulation while maintaining stable distribution patterns, providing a theoretical basis for nicotine regulation in flue-cured tobacco.