Commercial Eucalyptus plantations provide an important source of hardwood for forestry industries, worldwide. Several species of Mycosphaerella are associated with a destructive Eucalyptus leaf disease known as Mycosphaerella Leaf Blotch (MLB). During 2000, a survey was undertaken in several commercial Eucalyptus growing areas of South Africa to determine the identity of the Mycosphaerella spp. contributing to outbreaks of MLB. Symptomatic leaf samples were collected from three major Eucalyptus growing areas and the Mycosphaerella spp. were isolated. Isolates were identified using morphology, ascospore germination patterns and sequence data from the ribosomal DNA operon. Six species, namely M. ellipsoidea, M. irregulariramosa, M. juvenis, M. lateralis, M. marksii, M. nubilosa, as well as a new species, described here as M. fori sp. nov., were recognized. Mycosphaerella nubilosa was the most common species isolated from commercial plantations, particularly on E. nitens, and appears to be the dominant species contributing to MLB. Data obtained in this study show that MLB is caused by a complex of species contributing to disease outbreaks in South Africa.