Abstract: In order to enhance the resistance of flue-cured tobacco variety Zhongyan100 to root diseases and its leaf quality, a new variety Yunongxiang201 was bred from Zhongyan100 as the donor and selected by using EMS mutagenesis, glandular trichome density type detection and black shank disease nursery identification. The new variety was approved by the National Tobacco Variety Approval Committee in April 2023. Yunongxiang201 exhibited stable genetic traits, consistent and strong field growth, and was easy to cure. It had resistance to black shank (Race 1) and Fusarium root rot, moderate resistance to brown spot, moderate susceptibility to black shank disease (Race 0), root knot nematode disease and PVY, yet susceptibility to bacterial wilt, TMV and CMV, respectively. The comprehensive resistance of Yunongxiang201 was comparable to NC89, and its resistance to root disease was superior to Zhongyan100. Compared with NC89 and Zhongyan100, Yunongxiang201 had higher yield and output value, and better main economic traits. The variety meets the current quality requirements for raw material of cigarettes, with high appearance quality, coordinated chemical composition, and outstanding sensory evaluation of sweetness. It features uniformly matured leaves, thus is suitable for grain and tobacco fusion and mechanical harvest. Yunongxiang201 is proposed to be planted in Huanghuai tobacco area with irrigation conditions.