This study identified genes that distinguish Australian Fusarium oxysporum f.sp. vasinfectum (Fov) isolates from related co-localized non-pathogenic F. oxysporum isolates and from non-Australian Fov isolates. One gene is a homologue of the F. oxysporum f.sp. lycopersici (Fol) effector gene SIX6, encoding a 215-residue cysteine-rich secreted protein. The Six6 proteins from Fol and Fov contained eight conserved cysteine residues, five of which occurred in the highly diverged 48-amino-acid region where FovSix6 differs from FolSix6 at 32 residues. Two other potential effector genes, PEP1 and PEP2, were identified in a cDNA library of Fov genes expressed during infection of cotton. The presence of FovSIX6 and other differences in DNA fingerprints clearly distinguished Australian Fov isolates from non-Australian Fov isolates and these differences further support the hypothesis based on earlier phylogenetic analysis that Australian Fov is different from Fov in other cotton-growing areas. A specific diagnostic for Fov based on FovSIX6 is described.