Cotton (Gossypium hirsutum) is a billion-dollar crop in regional New South Wales (NSW) and Queensland, Australia. Fusarium wilt (FW) caused by Fusarium oxysporum f. sp. vasinfectum (Fov) is an economically important disease. Initial disease losses of up to 90% when the disease was first detected resulted in fields being taken out of cotton production. The disease is now well-managed due to the adoption of highly resistant varieties. However, annual disease surveys recently revealed that the disease dynamic has changed in the past few seasons. With relatively mild and wet weather conditions during the 2021/22 growing season, FW was detected in eight surveyed valleys in NSW and Queensland, with the disease incidence as high as 44.5% and 98.5% in individual fields in early and late seasons, respectively. Fov is genetically distinct and evolved from local Fusarium oxysporum strains. Additionally, the pathogen was reported to evolve rapidly under continuous cotton cropping pressure. However, our knowledge of the genetic composition of the prevailing population is limited. Sequences of the translation elongation factor alpha 1 (TEF1) revealed that 94% of Fusarium isolates recovered from FW-infected cotton were clustered together with known Australian Fov and relatively distant related to overseas Fov races. All these isolates, except for nine, were further confirmed positive with a specific marker based on the Secreted in Xylem 6 (SIX6) effector gene. Vegetative compatibility group (VCG) analyses of 166 arbitrarily selected isolates revealed a predominance of VCG01111. There was only one detection of VCG01112 in the Border Rivers valley where it was first described. In this study, the exotic Californian Fov race 4 strain was not detected using a specific marker based on the unique Tfo1 insertion in the phosphate (PHO) gene. This study indicated that the prevalence and abundance of Fov across NSW and Queensland in the past five seasons was probably independent of its genetic diversity.