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Genotyping studies of Australian Scedosporium isolates have revealed the strong prevalence of a recently described species: Scedosporium aurantiacum. In addition to occurring in the environment, this fungus is also known to colonise the respiratory tracts of cystic fibrosis (CF) patients. A high throughput Phenotype Microarray (PM) analysis using 94 assorted substrates (sugars, amino acids, hexose-acids and carboxylic acids) was carried out for four isolates exhibiting different levels of virulence, determined using a Galleria mellonella infection model. A significant difference was observed in the substrate utilisation patterns of strains displaying differential virulence. For example, certain sugars such as sucrose (saccharose) were utilised only by low virulence strains whereas some sugar derivatives such as D-turanose promoted respiration only in the more virulent strains. Strains with a higher level of virulence also displayed flexibility and metabolic adaptability at two different temperature conditions tested (28 and 37°C). Phenotype microarray data were integrated with the whole-genome sequence data of S. aurantiacum to reconstruct a pathway map for the metabolism of selected substrates to further elucidate differences between the strains.