The invention discloses molecular markers linked with a tobacco black shank-resisting Ph gene and application of the molecular markers. The serial numbers of the molecular markers are Scf_30K, TM62, ST141 and InDel0617, and the nucleotide sequences of amplification products of the molecular markers are shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4. The invention further discloses application of assistant selection of the molecular markers linked with the tobacco black shank-resisting Ph gene in a first hybridization generation, a second backcross generation and subsequent backcross thereof and a selfing improvement system (group) and application of assistant selection of the molecular markers linked with the tobacco black shank-resisting Ph gene in black shank resistance improvement. According to the molecular markers linked with the tobacco black shank-resisting Ph gene and the application of the molecular markers in improvement of the resistance of an existing good variety, a foundation is laid for fine location and gene clone of the tobacco black shank-resisting Ph gene, resistance screening can be performed on the tobacco black shank-resisting Ph gene in hybridization and backcross and subsequent generations thereof of a tobacco resistance improvement candidate material, the advantages of being convenient, fast, accurate, efficient and capable of presenting the codominance are achieved, and a convenient, fast, practical, scientific and efficient way is supplied to tobacco variety resistance improvement through marker-assisted selection (MAS).