Thielaviopsis basicola is a ubiquitous soil borne fungal plant pathogen with a wide host ran e. It is the causal agent of black root rot on many agriculturally important crops such as cotton, tobacco and legumes. Twenty-five T. basicola isolates collected from three cotton growing regions, and peat and lettuce soils from a range of locations were examined for genetic variation using the RAPD-PCR technique with 10 arbitrary primers. DNA polymorphisms were detected among isolates from the cotton-growing regions of Goondiwindi, Qld, and Narrabri and Warren, NSW. A phenogram was constructed using the unweighted pair-group method with arithmetic averages (UPGMA) for cluster analysis. Isolates from two cotton-growing regions each clustered into a distinct group based on RAPD-PCR profiles suggesting independent evolution of T. basicola between these regions. Isolates from the third cotton-growing region did not cluster and were distributed between the other two regions, suggesting migration and gene flow between these regions. Lettuce isolates clustered with peat isolates providing more evidence that peat is a source of T basicola found in lettuce soils. The results indicated that RAPD-PCR is a useful tool in detecting genetic variability in populations of T. basicola.