Abstract: To establish a molecular marker-assisted selection system for tobacco with resistance against TMV and promote the breeding of TMV resistant flue-cured tobacco varieties, K326 (quality but susceptibility to TMV)was used as recipient parent and crossed with CV87(resistance to TMV)as donor parent. Combining with molecular marker-assisted selection after multiple backcross and self-cross, TMV resistance gene N from CV87 was crossed into K326. The results showed that in 293 BC₂S₃ advanced generation materials (K326×CV87), there were 108 materials contained N gene, in which the N genotype of 107 materials was dominant homozygous because the P value of their resistance separation ratio was less than 0.05. Therefore, molecular marker-assisted selection has high reliability and can promote the breeding efficiency of TMV-resistant tobacco varieties.