Abstract: To breed tobacco germplasm with double resistance to Phytophthora nicotianae and tobacco mosaic virus (TMV), haploids were obtained from the crossing of Nicotiana africana and F₁ hybrids between Coker371 and Coker176 through maternal-derived haploid technology. Maternal haploid seedlings were rapidly identified by morphological observation and flow cytometry. After molecular marker assistant selection, the target maternal haploid was doubled via leaf midvein culture, the germplasm line RBST (Resistant to Black Shank and TMV) with double resistance against Phytophthora nicotianae and TMV was developed. Resistance identification with artificial inoculation in seedling stage, molecular marker assisted detection and field agronomic trait identification showed that with stable resistances and agronomic traits, RBST could be used as the material of resistance source for the oriented improvement of resistances in tobacco.