Fusarium wilt of banana, caused by Fusarium oxysporum f. sp. cubense (Foc), is regarded as one of the most devastating diseases. Cavendish cultivars (AAA genome) are highly susceptible to race 4 of the pathogen. The regulation of defence-related genes in ‘GCTCV-218’, a Fusarium wilt-tolerant Cavendish selection, was studied. A cDNA library, enriched for genes up-regulated in ‘GCTCV-218’ after Foc infection when compared to susceptible Cavendish cultivar ‘Williams’, was previously constructed using suppression subtractive hybridisation (SSH) and screened using DNA micro-array technology. Potentially induced clones (334) were selected and sequenced. Four defence-associated genes (catalase 2, pectin acetyl esterase (PAE), PR-1 and PR-3) were selected for expression profile analysis using real-time reverse transcriptase PCR (RT-PCR). ‘GCTCV-218’ showed significantly lower disease severity and incidence than the susceptible ‘Williams’ in replicated greenhouse and field trials. Several defence-related transcripts were identified by BLASTX searches, including genes coding for PR-1, PAE, xylanase inhibitor, peroxidase, catalase 2, metallothionein, response regulator 6 and tripsin inhibitor. Inverse Northern dot blot data indicated that all of the above-mentioned transcripts were up-regulated in ‘GCTCV-218’ 6 hours post inoculation (hpi) compared to ‘Williams’. Quantitative RT-PCR results confirmed that all four genes were differentially expressed in ‘GCTCV-218’ 3 and 6 hpi, confirming SSH results. PR-1 and PAE were induced very early (3 hpi) in ‘GCTCV-218’, while PR-3 and catalase 2 followed with a significant induction 6 hpi. This study concluded that ‘GCTCV-218’ is able to respond rapidly to Foc infection by activating both a biochemical and structural defence mechanism. The tolerance of ‘GCTCV-218’ was further linked to a significant increase in the induction cell wall-associated phenolic compounds.