Abstract: To identify the primary pathogens causing Fusarium root rot in tobacco-growing regions of Henan Province, and to provide a theoretical basis for developing precise and efficient control strategies. A total of 291 purified strains were isolated from tobacco root rot plants collected from different tobacco-growing regions in Henan Province using tissue isolation methods. Identification was based on morphological characteristics, construction of monophyletic phylogenetic trees based on different gene fragments (rDNA-ITS, tef1-α, and RPB2 genes), and indoor pathology tests involving artificial inoculation at the stem base. The results revealed that 231 strains exhibited typical Fusarium morphological characteristics. Phylogenetic tree analysis identified 139 strains as Fusarium oxysporum, 61 strains were N. solani, 14 strains were F. proliferatum, 10 strains were F. equiseti, 1 strain was F. ipomoeae, 1 strain was F. chlamydosporum, 3 strains of F. falciforme, 1 strain of F. verticillioides, and 1 strain of F. delphinoides. In Henan's tobacco-growing regions, Fusarium root rot exhibits a pattern of mixed infections involving multiple pathogens, with the dominant species F. oxysporum and Fusarium solani accounting for 60.17% and 26.41% of cases, respectively, for a combined total of 86.58%. This study identified the primary pathogen spectrum of tobacco root rot in Henan's tobacco-growing regions, confirming nine Fusarium species. F. oxysporum and N. solani accounted for 86.58% of infections, forming the dominant pathogen group. The first detection of F. delphinoides in this region indicates that root rot pathogens in Henan tobacco-growing areas exhibit diversity and mixed infection characteristics. This study provides reliable evidence for formulating targeted control strategies.