Abstract: To improve resistance to tobacco virus in K326, a new flue-cured tobacco cultivar Zhongyan300 was developed by crossing recurrent parent K326 and three donor parent cultivars with resistance to virus using classical breeding selection and marker assisted selection (MAS) methods. The new variety was certified by the National Tobacco Variety Examination and Approval Committee in December, 2018. The results of variety experiments showed that the main economic and agricultural characters of Zhongyan300 were in accordance with K326. The results of industry evaluation showed that appearance quality, physical properties, chemical composition and sensory quality evaluation of Zhangyan300 were also in accordance with K326. As for resistance to diseases, Zhongyan300 showed higher resistance to viral diseases than K326, being immunized with tobacco mosaic virus (TMV), resistant to cucumber mosaic virus (CMV) and potato virus Y (PVY). In summary, the new flue-cured tobacco cultivar Zhongyan300 keeps the excellent characteristics of cultivar K326. Meanwhile, resistance to viral diseases was significantly improved.