Cytoplasmic male sterility, (CMS), in tobacco is of great importance for the heterosis breeding, saving the time-consuming process for emasculation. Crossing the wild Nicotiana knightiana with N. tabacum/cv. Djbec 44, seeds of good germinating ability were obtained. The cultivated F1 plants showed complete pollen sterility. After pollination of F1 plants with pollen from N. tabacum/cv. Djbec 44, seed capsules were formed, which showed that the female gametophyte was normal. In BC1P2 progeny male sterile plants were found. They possessed normally developed corolla, pistils with two locules and stamens with normal anther morphology and filament size. As a result of cytological investigation it was established that the stamens had lost the function of male reproductive organs. The male sterility of BC1P2/N. knightiana x N. tabacum/x N. tabacum was preserved in the BC2-BC6 progenies, which confirmed its cytoplasmic origin.