Abstract: Cytoplasmic male sterile line (CMS line) is an effective genetic tool for tobacco hybrid breeding. At present, there is a single type of CMS used in tobacco hybrid breeding in China. In order to identify the similarities and differences between the male sterile natural mutant SK01 and the sterile cytoplasm used in production, and to analyze its application potential in tobacco breeding and production, tab1-CMS sterile line SK01 and its homokaryon heterogeneous line K326 were used as the female parents in crossing with four flue-cured tobacco varieties to construct two sets of F1 hybrids. Eight F1 lines, SK01 and K326 were compared in two locations in the field or in artificial climate rooms. The main agronomic traits and disease resistance differences between tab1-CMS-specific DNA marker screening were carried out. The results showed that the SK01 cytoplasm enhanced the resistance of tobacco hybrid F1 to CMV and black shank, and had no significant cytoplasmic effect on resistance to PVY and TMV, plant height, stem circumference or other agronomic traits. A 588 bp specific fragment was amplified from the trnC-trnD gene interval of the chloroplast genome, to that can effectively distinguish the sterile cytoplasm of tobacco. It shows that the sterile cytoplasmic tab1-CMS identified in this study is significantly different from the sua-CMS type sterile line used in production and has no obvious adverse effects on agronomic traits and disease resistance. It is a new type of cytoplasmicsterility with certain application potential.