Mycosphaerella contains some of the most damaging foliar pathogens of Eucalyptus. Identification of the species has relied mainly on characters such as ascospore morphology and the mode of ascospore germination. Wide variation and overlap in morphological characters, especially ascospore shape and size, has lead to confusion in species concepts. This study examines the potential of RAPD–PCR to differentiate Mycosphaerella species that cause leaf diseases of Eucalyptus in Australia. RAPD–PCR was conducted on 39 isolates representing 10 species of Mycosphaerella collected from southern Australia. Primers were chosen mainly to distinguish between the two most common and damaging species in Australia, M. cryptica and M. nubilosa. The RAPD analysis revealed five groups within the 39 isolates, four of which represent distinct species: M. cryptica, M. gregaria, M. nubilosa and M. marksii. For these four species, the cluster analysis and RAPD patterns gave results that fitted well with classical taxonomic criteria. Variation within M. cryptica, M. nubilosa and M. marksii was low, even though the isolates within these species originated from diverse hosts and locations. The remaining species in this study grouped together and could not be easily separated using the RAPD data here.