Fusarium oxysporum f. sp. cubense (Foc) is a fungus that infects banana roots and causes a destructive plant disease called Fusarium wilt. Foc consists of three pathogenic races (Foc races 1, 2 and 4), classified according to their selective impairment of banana cultivars. Foc race 4 is economically important as it comprises strains that infect Cavendish bananas, which are the most widely planted variety of bananas in the world, in both the tropics (Foc TR4) and subtropics (Foc STR4). The aim of this study was to investigate which genes are potentially involved in fungal pathogenicity by comparing transcript-derived cDNA fragments (TDFs) from Foc STR4 and TR4 to those from non-pathogenic F. oxysporum using cDNA-AFLP analysis. This comparison resulted in the identification of 229 unique gene fragments which include the putative pathogenicity-related TDFs encoding chitinase class V (chsV), GTPase activating protein, Major Facilitator Superfamily (MFS) multidrug transporter and serine/threonine protein kinase (ste12) genes. Quantitative analysis of transcript abundance showed a significant increase in expression of chsV, MFS multidrug transporter and ste12 genes in Foc STR4 and TR4 compared with the non-pathogenic F. oxysporum. These genes play a role in escaping host defence responses and in cell wall degradation. In addition, pathogenicity-related genes from other formae speciales of F. oxysporum, such as the sucrose non-fermenting, cytochrome P450 and F-box protein required for pathogenicity genes, were significantly up-regulated in Foc STR4 and TR4 but not in F. oxysporum isolates non-pathogenic to banana. This study provides the first in vitro comparative analysis of TDFs expressed in pathogenic Foc race 4 isolates and non-pathogenic F. oxysporum isolates from banana.