Cotton is a billion-dollar crop in Australia, especially in the regional areas of New South Wales (NSW) and Queensland. Fusarium wilt caused by Fusarium oxysporum f. sp. vasinfectum (Fov) is of an important disease on cotton globally and in Australia. The Australian cotton industry is currently adopting highly resistant cultivars to Fov. Therefore, it is expected that Fusarium wilt pressure will be minimal on the cotton crop. However, we recovered a total of 186 putative Fusarium isolates from cotton seedlings exhibiting atypical symptoms of either Fusarium damping-off or Fusarium wilt sampled across NSW during the 2017/18 and 2018/19 seasons. On a basis of sequence analysis of the translation elongation factor 1-alpha (TEF1), eight Fusarium species were identified that being Fusarium oxysporum and F. equiseti species complex, F. falciforme, F. nygamai, F. brachygibbosum, F. acuminatum, F. chlamydosporum, and F. redolens. Of these, F. oxysporum species complex (FOSC) accounted for over 80% of the total number of isolates recovered. The predominant number of isolates (78.5%) were clustered with non-Australian Fov races 1–8. The remaining 21.5% of the FOSC isolates in our study was clustered with the Australian Fov biotypes, a unique phylogenetic group within the FOSC. Our study is the first TEF1-based assessment of Fusarium diversity associated with collar rot diseased cotton seedlings sampled across NSW, Australia.