Abstract: Fusarium solani is the dominant pathogen of tobacco Fusarium root rot. In order to clarify the pathogenicity differentiation of Fusarium solani in tobacco growing area of Henan Province, and establish a rapid and accurate detection method for F. solani in tobacco and soil, the pathogenicity of 162 F. solani strains from different growing area was analyzed by spore suspension root irrigation method. Based on the whole genome sequence of F. solani, specific primer pairs were designed and verified for its specificity and sensitivity to establish a PCR detection system for F. solani. The results showed that the tested strains exhibited significant differentiation in pathogenicity. Among them, strong virulence, moderate virulence, weak virulence and non-virulence strains accounted for 21.61%, 15.43%, 43.21% and 19.75%, respectively. Although strains with different virulence showed no significant differences in colony morphology, geographical origin analysis revealed that the disease index (DI) caused by strong virulence strains from tobacco-growing areas of central and western Henan was significantly higher than that in southern Henan tobacco-growing areas. The primer pair 803-1F/1R that designed and screened based on genomic sequences exhibited high specificity, which could specifically amplify the target fragment of approximately 294 bp, and the expression level of this specific band differed significantly among strains with different virulence. The optimized PCR detection system achieved a sensitivity of 100 pg/μL, enabling accurate and specific identification of Fusarium solani in inoculated diseased plants and their rhizosphere soil. Field plant tissue and soil sample detection results showed that the detection efficiency of this PCR system ranged from 80.00% to 92.31%, which is reliable, rapid and simple compared with traditional methods. This study confirmed that obvious intraspecific virulence differentiation exists in F. solani populations in tobacco-growing areas of Henan Province, with no direct correlation to geographical distribution. These findings provide an important basis for disease-resistant variety breeding and disease epidemic prediction. Moreover, the established PCR detection system can serve as an effective technical support for early diagnosis and monitoring of tobacco root rot caused by F. solani.