Abstract: Originated from the wild tobacco N. plumbaginifolia, the Ph gene is a high-quality resistance source to black shank in tobacco breeding. In order to develop SNP markers linked to this gene to meet the demand of current molecular breeding, this study used black shank-resistant flue-cured tobacco variety Coker371-Gold with the Ph gene and a susceptible variety Zhongyan texiang 301 without the Ph gene as materials. Using tobacco SSR markers and SNP variations of 33 cultivated tobacco germplasm resources based on whole genome resequencing, the molecular characteristics of the chromosomal fragment of N. plumbaginifolia carrying the Ph gene was described. By constructing a backcross population segregating for black shank resistance, an integrated genetic map of 8 known molecular markers linked to the Ph gene was drawn, and on the basis, more closely linked SNP markers to the Ph gene were developed. The results showed that: (1) the N.plumbaginifolia chromosomal fragment carrying the Ph gene was probably transferred into cultivated tobacco in the form of terminal translocation, and had a good homology relationship with the replaced cultivated tobacco chromosomal fragment, and its translocation site was located near 18.54 Mb of Yunyan 87 HIC_ASM_14 chromosome; (2) Ph gene was located at the end of the translocation fragment, which probably being close to the translocation site, and one closely linked specific SNP marker was obtained on each side of the gene. These results provided a theoretical basis for further creating a more valuable intercalary translocation line with small fragment, thereby providing technical support for better utilization of this black shank disease resistance source in the future.