The ascomycete Sclerotinia sclerotiorum is a plant pathogen with a very broad host range. In order to identify and characterize genes involved in S. sclerotiorum infection of Brassica napus (canola), expressed sequence tags (ESTs) were examined from libraries prepared from three tissues: complex appressorium (infection cushions), mycelia grown on agar and lesions formed on leaves of B. napus. A high proportion of genes (68%) had not been previously reported for S. sclerotiorum in public gene or EST databases. The types of novel genes identified in the infection cushion library highlights the functional specificity of these structures and similarities to appressoria in other fungal pathogens. Quantitative real-time PCR was used to analyse tissue specificity and timing of transcription of genes with best matches to MAS3 (appressoria-associated protein from Magnaporthe grisea), cellobiohydrolase I, oxaloacetate acetylhydrolase, metallothionein, pisatin demethylase, and an unknown gene with orthologs in fungal pathogens but not in saprophytic fungi.