Cytoplasmic male sterile (CMS) lines are valuable resources in plant breeding for heterosis utilization, guaranteeing quality of hybrid seeds, and genetic integrity. Nta(sua)S K326, which contains the nuclei of Nicotiana tabacum cv. K326 and cytoplasm of N. suaveolens, is currently the dominant CMS source used in tobacco leaf production in Yunnan, China. Therefore, the alternative CMS lines are required. Here, the Nta(gla)S K326 with cytoplasm of N. glauca has been generated from asymmetric somatic hybridization between protoplasts of K326 and N. glauca followed by backcrossing with K326. Flow cytometry analysis revealed that Nta(gla)S has almost identical ploidy level as that in K326. Moreover, Nta(gla)S, Nta(sua)S, and K326 plants showed similar relative growth rate, yield, and contents of several chemical components in the field. Further scaning electron microscopic observation of floral organs revealed that the Nta(gla)S has shortened filaments and shrivelled stamens, whereas the Nta(sua)S has severe degenerated and carpel-liked stamens, compared with fertile K326 plants. In addition, the expression patterns of several mitochondrial genes in Nta(gla)S, Nta(sua)S, and K326 plants were compared. Furthermore, the cytoplasm source of Nta(gla)S and Nta(sua)S could be distinguished by cleaved amplified polymorphic sequence (CAPS) analysis with chloroplast-specific universal primers. Taken together, these results showed the development of a Nicotiana CMS hybrid via asymmetric somatic hybridization, suggesting that flow cytometry analysis at early stage could facilitate and accelerate the selection process of generating CMS lines. Importantly, the Nta(gla)S provides a valuable germplasm as an alternative CMS line instead of Nta(sua)S in Nicotiana hybrid breeding.