The introduction of hybrid crop varieties has enabled spectacular growth in productivity owing to hybrid vigor and increased uniformity. To produce hybrid seeds, a pollination control system is required to prevent unwanted self-pollination. New pollination control systems have been developed with the aid of cytoplasmic male sterility (CMS), defined as the inability to produce functional pollen. With the use of CMS the emasculation, which is laborious process in plants with hermaphrodite flowers, is eliminated. In tobacco CMS can be generated in interspecific hybrids by the combined action of the nucleus of N. tabacum and the cytoplasm genes of wild species. In our study CMS sources with cytoplasms from N. velutina (vel), N. maritima (mar), N. amplexicaulis (amp), N. paniculata (pan), N. longiflora (lon), N. benthamiana (ben), N. rustica (rus), N. excelsior (exc) and N. africana (afr) are recurrently backcrossed with cv. Coker 254 to developed CMS analogues. All these cytoplasms produce complete male sterility in cv. Coker 254. The obtained CMS analogues are included as female components and Line 285 of tobacco as male parent in order to create male sterile F1 hybrids. Fertile hybrid from the combination cv. Coker 254 x Line 285 is used as a control. Comparative morphological characterization of CMS F1 hybrids as well as of the fertile F1 hybrid is carried out. The agronomic performance of five CMS sources (vel), (mar), (amp), (pan), and (lon) in the F1 hybrids exceed the control in respect to plant height, leaf number, area and yield. Some depressing effect on these features is observed in CMS hybrids with cytoplasms (exc) and (afr). The rest CMS hybrids (ben) and (rus) have similar agronomic characteristic as the fertile F1 hybrid. In conclusion the CMS F1 hybrids Coker 254 x Line 285 with cytoplasms from N. velutina, N. maritima, N. amplexicaulis, N. paniculata and N. longiflora are defined as most suitable in tobacco breeding. These CMS cytoplasms have normal female gametophyte and good seed productivity.