Abstract: It is difficult to distinguish Fusarium oxysporum from other Fusarium species using traditional morphological observations or molecular analysis based on rDNA-ITS sequencing. In order to accurately identify F. oxysporumin legumes, a pair of primers named P₁/P₂ was designed based on differences in ribosomal DNA intergenic spacer (rDNA-IGS) sequences of the Fusarium genus, which can be used to amplify DNA from F. oxysporum by conventional PCR. More than 23 species of root rot pathogens were used to verify the specificity of the primers. P₁/P₂ could amplify a unique 1081 bp sequence from different biotypes of F. oxysporum while it could not amplify from other root rot pathogens. The sensitivity of P₁/P₂ was 100 pg for genomic DNA and 100 conidia/g soil for the root rot pathogens. Furthermore, this pair of primers could directly amplify sequences from the genomic DNA of F. oxysporum diseased plant samples without pathogen isolation, indicating that this is a rapid and effective legume root rot pathogen detection technology.