A DNA-based macroarray was developed to quickly and accurately identify all known races and the two Australian biotypes of Fusarium oxysporum f. sp. vasinfectum, the causal agent of Fusarium wilt of cotton. The macroarray utilized oligonucleotide probes designed from sequences of the elongation factor gene unique to Races 1, 3, 4, 8 or the Australian biotypes. Starting with diseased tissue, the entire detection process from DNA extraction to race identification could be completed within 8 hours. The assay reproducibly identified all F. oxysporum f. sp. vasinfectum races and biotypes from cultures, and greenhouse- or field-grown infected cotton plants.