Alternaria core rot of red apple cultivars is a serious post-harvest disease in South Africa. Thirty isolates of Alternaria spp. previously isolated from apple, together with reference isolates of A. alternata and A. infectoria, were characterised and grouped according to their sporulation patterns and conidial morphology. Isolates were identified as belonging to A. arborescens, A. infectoria and A. tenuissima species-groups. The isolates were also analysed for production of mycotoxins and other secondary metabolites and their cultural characteristics on DRYES medium were recorded. Apple fruit were artificially inoculated with the thirty-two Alternaria isolates and the resulting lesion types were recorded. A data matrix was constructed using all these characters and subjected to cluster analysis to show the similarity between different isolates. Isolates classified as A. infectoria species-group based on sporulation patterns, cultural and biochemical data could be easily differentiated from isolates classified as A. arborescens and A. tenuissima species-groups, which clustered close together. Isolates were further subjected to DNA sequence analysis of the internal transcribed spacers (ITS) 1 and 2 of the nuclear rRNA gene. A phylogeny estimated from the ITS data set delineated two clades, one being typified by the A. infectoria species-group, and the other representing both A. tenuissima and A. arborescens species-groups. The ITS data set clearly separated isolates of A. infectoria species-group from the other species-groups, as all isolates had a distinction of 35 base pair insertions and 6 base pair deletions in the ITS regions. The results obtained in the present study showed that the major pathogens associated with core rot disease of Top Red apples in South Africa belong to the A. tenuissima species-group.