Phomopsis husk rot, an emerging disease in macadamia, is characterised by diffuse, soft, and spongy black lesions appearing on macadamia fruit pericarp. Phomopsis husk rot causes premature fruit drop and a reduction in kernel quality. Recent studies have indicated that species within the genus Diaporthe cause the disease, and that damage to the pericarp is a prerequisite for infection. In order to better understand disease aetiology, a thorough taxonomic analysis of the isolates of Diaporthe spp. from diseased macadamia pericarps is required. Success in the delimitation of species within this genus has been attributed to multi-locus DNA sequence analyses of several specific loci. The rDNA internal transcribed spacer (ITS) and beta-tubulin (TUB) regions represent the most promising targets for inferring meaningful taxonomic and phylogenetic relationships for species in the genus Diaporthe. In this study, we used ITS and TUB regions of isolates obtained from different macadamia cultivars in Australia, to characterise the Diaporthe spp. that cause Phomopsis husk rot. Pathogenicity assays with different Diaporthe spp. were used to assess pathogenic variation and virulence on macadamia varieties. Preliminary results indicate significant variation exists between cultivars to infection by Diaporthe spp. A more exhaustive range of cultivars are now being studied to elucidate any differences in susceptibility between cultivars and gain a temporal understanding of the infection process. The study revealed new information on varietal susceptibility, pathogenic variation and virulence of species of Diaporthe on macadamia. The scientific understanding of the genus Diaporthe, as well as our understanding of the aetiology of Phomopsis husk rot, underpin the development of future control strategies for the disease, leading to greater yields and higher profitability for farmers.