Fusarium isolates were extracted from stems and rhizosphere soils of 79 populations of four Gossypium species native to two regions of inland Australia in 2001. Six Fusarium species were isolated from 31 (3%) of the 919 stem samples. F. semitectum was predominant, occurring mostly in G. bickii populations and accounting for 81% of the isolates recovered. Twenty Fusarium species were isolated from 68 (87%) of the 78 composite soil samples, with F. solani (71%), F. compactum (8%), F. oxysporum (5%), F. graminearum (3%), and F. crookwellense (3%) being the most prevalent. Significant differences in the relative densities indicated that F. solani was more common in the soil from G. sturtianum populations than from G. bickii populations, and that F. crookwellense was more common in South Australian soils than in Queensland and Northern Territory soils. Fusarium oxysporum had a relatively greater relative density in the rhizosphere soils of G. australe plants than in that of sympatrically growing G. sturtianum plants. Fifteen (17%) of the 89 F. oxysporum isolates produced typical Fusarium wilt symptoms on cultivated cotton (G. hirsutum cv. ‘Siokra 1–4’), therefore they were classified as wild Fov. Soil samples collected from G. sturtianum populations in the Arkaroola-Leigh Creek region in South Australia had the highest incidence of wild Fov (24%). Two wild Fov isolates were similar in virulence to a cotton field Fov isolate in the glasshouse experiments, indicating that they could incite Fusarium wilt disease in cotton fields. The presence of wild Fov in native Gossypium populations suggests that the Fov occurring in Australian cotton fields may be of indigenous origins.