Strains of the fungal wilt pathogen Verticillium dahliae cause considerable economic losses in field crops throughout the world. We have investigated the use of molecular genetic techniques for the identification and classification of strains infecting cotton plants (Gossypium spp.) in the major production regions of Australia. The amplification, sequencing, and restriction digestion of specific ribosomal DNA sequences proved to be diagnostic for the genus/species, but was incapable of differentiating between individual isolates with quite diverse morphologies or origins. RAPD-PCR analysis using a subset of 13 informative decamer primers, however, did reveal significant differences between the isolates and enabled the genetic similarity of the V. dahliae strains to be estimated. RAPD-PCR fingerprints were shown to be reproducible following in planta culture of V. dahliae isolates and could be a valuable tool for the identification and epidemiological study of fungal populations. Although fungal isolates were found to elicit a range of reactions during pathogenicity testing on 5 cotton cultivars, comparison of the RAPD-PCR banding patterns did not suggest a strong correlation between molecular genetic and virulence characteristics.