Fusarium wilt of cotton has emerged as a major threat to cotton production, since it was first recorded in Australia in 1993. Control of Fusarium wilt depends on understanding genetic diversity within the fungus that causes the disease, Fusarium oxysporum f.sp vasinfectum (Fov). We have analysed genetic diversity within Fov by DNA amplification fingerprinting (DAF), and restriction fragment length polymorphism (RFLP) and sequence analysis of the intergenic spacer (IGS) region of the ribosomal (r) DNA. The Australian isolates of Fov were compared to overseas isolates that represented races I, 2, 3, 4, 5, 7, 8 and A of the pathogen, and also to other formae speciales of F. oxysporum and species of Fusarium. We have identified two distinct genotypes amongst the Australian isolates of Fov that correspond with vegetative compatibility groups (VCGs) 01111 and 01112. We are using the information we have obtained on genetic diversity among Australian isolates of Fov to develop a polymerase chain reaction (PCR)-based DNA diagnostic system for the rapid detection of Fov directly from infected plants and seed, and infested soil. This diagnostic test will be an invaluable tool for the cotton industry, as early detection of Fusarium wilt is critical to the containment and control of the disease