Abstract: In recent years, newly emerging root and stem diseases have been observed in certain major tobacco-growing areas of Xiangxi Prefecture, Hunan Province, and are showing a spreading trend. Therefore, there is an urgent need for accurate identification of the new diseases and precise characterization of their causal agents. To achieve this, we conducted field observations of typical symptoms, pathogen isolation, morphological characterization, Koch’s postulates verification, molecular identification, and phylogenetic analysis. The results showed as follows. The newly occurred Fusarium root rot in Baojing County is caused by Fusarium oxysporum and F. solani, with F. oxysporum as the predominant pathogen. The root and stem disease causing "black spots" on tobacco roots/stems and aboveground growth retardation in Yongshun County is attributed to Rhizoctonia solani Kühn AG-4 HGI anastomosis group; this disease leads to poor tobacco growth and severe yield loss, and the pathogen is pathogenic to corn, soybean, chickpea, and cotton plants under artificial inoculation conditions. The pathogenic bacterium Ralstonia solanacearum isolated from Baojing, Huayuan, and Longshan Counties was identified as Phylotype Ⅰ, with Sequevar 17 (Huayuan) and Sequevar 44 (Baojing and Longshan). Notably, Sequevar 44 is the first report in this region. This study provides a scientific basis for the accurate diagnosis and control of the newly emerging root and stem diseases of flue-cured tobacco in Xiangxi Prefecture, as well as for crop selection under the new mode of grain-tobacco integrated planting.