Abstract: Aiming to identify the pathogen types of tobacco root rot disease in Chenzhou and Yongzhou regions of Hunan Province, the effective control agents were screened for the main pathogens, samples of tobacco root rot disease and rhizosphere soil were collected from the main tobacco-growing area and isolated by using tissue separation method and plate dilution method, respectively. The pathogen types were determined by morphological observation, EF-1α sequence phylogenetic analysis, and pathogenicity assays. The growth rate method was employed to determine inhibitory effect of the tested fungicides on mycelia growth of the main pathogenic Fusarium species. The results showed that 128 fungal strains was isolated, including 87 Fusarium strains, which were divided into Fusarium commune, Fusarium incarnatum, Fusarium fujikuroi, Fusarium solani and Fusarium proliferatum, Fusarium falciforme, Fusarium humulium. Among them, F. commune accounted for the highest proportion of 39.08%, followed by F. solani and F. incarnatum, both with the proportions of 14.94%. Virulence tests showed that these Fusarium strains could cause tobacco root rot disease and were identified as the pathogen of this disease. F. commune was sensitive to 40% Prochloraz EW, 43g/L Tebuconazole SC and 15% cyanomethacrylate SC with EC₅₀ of 0.026, 0.079 μg/mL and 0.169 μg/mL, respectively. The results of pot experiment indicated that 40% Prochloraz EW exhibited the most significant control effect, with relative control effect of 94.43%±2.79%, followed by 43 g/L Tebuconazole SC and 15% Phenamacril SC, with the control efficiency of 88.63%±1.68% and 83.07%±2.55%, respectively. This study demonstrates that Fusarium commune is the main pathogen of tobacco root rot disease in Chenzhou and Yongzhou of Hunan Province. Among the tested agents, 40% Prochloraz EW proves the best control effect on this pathogen.