Fusarium wilt of Proteaceae was first reported in 1998 on the genus Protea in the North West province of South Africa and in Zimbabwe. Subsequently it has been reported from the Canary Islands (Tenerife), Chile and Portugal and might also be a potential risk in Australia. Fusarium oxysporum, the causal agent of Fusarium wilt, blocks the vascular tissue of the host plant, thereby causing wilt, chlorosis and plant death. In this study, molecular techniques have been employed to characterise the pathogen, which has only been morphologically identified thus far. Fusarium oxysporum isolates were obtained from Agathosma, Leucadendron, Leucospermum, Protea and Lavandula originating from South Africa, Zimbabwe and Portugal. Partial sequences of the translation elongation factor 1 alpha (EF-1 alpha) gene and the mitochondrial small subunit (mtSSU) region were generated. Phylogenetic analysis of the sequence data confirmed all the isolates to be Fusarium oxysporum. Fusarium oxysporum isolates from a specific host did not cluster together, indicating that isolates from the same host are not clonal. The DNA phylogenies did not follow the formae speciales classification system.