The disease- and aphid-resistant species, N. gossei (2n=36), was crossed with N. tabacum cv. Kroumovgrad 90, an oriental tobacco type. The F₁ hybrids from this cross were all sterile. Regenerates of this cross were developed by in vitro methods, and they were partially fertile. The F₁ hybrids, their regenerates, and seed progenies were evaluated for resistance to tobacco aphids, powdery mildew, and tobacco mosaic virus by artificial inoculation in greenhouse conditions. The inheritance of resistance to aphids, powdery mildew, and TMV was dominant in F₁ hybrids. Resistance to aphids, blue mold, and powdery mildew was also expressed in the fertile regenerates, and in the first and second seed progenies. These regenerates are of great interest for the genetics and breeding of tobacco.