Abstract: To identify the pathogens causing root rot disease in South Anhui tobacco-growing area and to screen novel, highly efficient fungicides against the dominant pathogens, pathogen isolation from tobacco root rot samples was carried out by tissue separation method, the pathogen types were determined by morphological and molecular biological methods, the antifungal activity of five fungicides against the pathogens was measured using mycelial growth rate method, and their control efficacy was verified in pot experiments. A total of 92 strains of Fusarium were isolated, including F. asiaticum, F. solani, F. commune, F. oxysporum and F. equiseti. Among them, F. asiaticum and F. solani were the dominant species, accounting for 34.78% and 30.43%, respectively. Pathogenicity tests revealed that all five Fusarium species were pathogenic. The pathogenicity of F. asiaticum on tobacco was reported for the first time. Virulence assays showed that 200 g/L pydiflumetofen·difenoconazole SC, 450 g/L cyclobutrifluram SC, and 30% trifloxystrobin·tebuconazole SC had relatively strong virulence against the five Fusarium species, with EC₅₀ values ranging from 0.001-4.8 μL/L. The pot experiment showed that 450 g/L cyclobutrifluram exhibited the best control efficacy, with a relative control efficacy of (82.30% ± 1.93) %. This study identified the main pathogenic species causing root rot in South Anhui tobacco-growing area, screened for highly effective fungicides, and provided a theoretical basis for the scientific prevention and control of tobacco Fusarium root rot.