Abstract: To construct a tobacco whole-genome module library and carry out tobacco molecular module breeding, in this study we used K326 as the recurrent parent and flue-cured tobacco OX2028 and aromatic tobacco Samsun as donor parents. After hybridization, continuous multi-generation backcrossing, and selfing, two sets of molecular module libraries covering the entire genome of the donor parents were obtained with K326 as the background. The genome of the molecular module mostly returned to the recurrent parent K326, and field agronomic and disease resistance traits were biased towards the parent K326. However, there was still a wide range of genetic variation. Among them, the coefficient of variation for waist leaf length was the smallest for two consecutive years, and the coefficient of variation for internode length was the largest for two consecutive years. The basic agronomic traits of the population showed a continuous normal or near-normal distribution, which is consistent with the phenotypic distribution characteristics of quantitative traits. Seven molecular modules with significant changes in important traits were identified. Compared to K326, 32 materials with significantly improved upper leaf opening, 25 materials with significantly increased leaf number, 111, 25, and 5 materials with significantly increased resistance to TMV, CMV, and PVY virus diseases, and 44 and 52 materials with significantly increased resistance to black shank and bacterial wilt diseases were identified. The tobacco molecular module library constructed in this study can be used for subsequent QTL mapping and gene function research on important tobacco traits.