Plants have developed complex defence mechanisms to protect themselves against pathogens. A wide-host range fungus, Austropuccinia psidii, which has caused severe damage to ecosystems and plantations worldwide, is a major threat to Australian ecosystems dominated by Myrtaceae. In particular, the east coast wetland foundation species Melaleuca quinquenervia appears to be variably susceptible to this pathogen. Understanding the molecular basis of host resistance would enable better management of this rust disease. We identified resistant and susceptible individuals of M. quinquenervia, and explored their differential gene expression in order to discover the molecular basis of resistance against A. psidii. Rust screening of germplasm showed a varying degree of response with totally resistant to highly susceptible individuals. We used transcriptome profiling in samples collected before and five days after inoculation (dpi). Differential gene expression analysis showed numerous defence-related genes induced in susceptible plants at 5 dpi. Mapping reads against the A. psidii genome showed that only susceptible plants contained fungal-derived transcripts such as ‘planta-induced rust gene’. Resistant plants exhibited an over-expression of candidate A. psidii defence-related genes such as receptor-like kinases (RLK) and nucleotide-binding site-leucine-rich repeat (NBS-LRR) proteins, as well as transcriptional regulators and PR-proteins. We identified large differences in gene expression of defence-related genes between biological replicates of resistant plants.