Cotton (Gossypium L.) originates from dicotyledonous, perennial shrubs cultivated for their soft fibers. A select group of species, collectively known as upland cotton within the Gossypium genus, is grown globally as annual crops and accounts for 90% of worldwide cotton production. In Australia, soilborne diseases such as Fusarium wilt, Verticillium wilt, and black root rot have caused substantial reductions in cotton yields. This project, facilitated by the ARC Hub for Crop Protection, aims to deepen our understanding of these pathosystems by characterising the underlying pathogens—Fusarium oxysporum f. sp. vasinfectum (Fov), Verticillium dahliae, and Berkeleyomyces rouxiae—with respect to their virulence, host specificity, and localisation. Fluorescent protein-tagged defoliating (VCG 1A) and non-defoliating (VCG 2A) pathotypes of V. dahliae were observed to traverse plant vascular systems, inducing wilt symptoms and endophytically colonising common weed species native to Australian landscapes. This finding suggests that weed plants may serve as reservoirs for these pathogens, facilitating infections in subsequent cropping cycles. Conversely, symptoms caused by B. rouxiae, such as reduced plant vigour, stunted growth, and black lesions on stems and roots, are predominantly expressed during early seedling establishment, whereas the wilt symptoms induced by Fov are more variable and typically manifest in the later stages of plant development. A rapid screening method was employed to assess the virulence of B. rouxiae and F. oxysporum strains on cotton plants, revealing significant intra-specific variations in their ability to cause diseases on cotton plants. Notably, certain F. oxysporum strains that did not phylogenetically align with known Fov races were capable of colonising cotton and exhibited mild virulence on these plants. This finding suggests a potential role for these strains in the evolution of Fov populations in the field. Lastly, we represent long-read genome assemblies for representatives of these pathogen strains, aiming to provide genomic resources that support the identification of effector genes associated with virulence and the development of diagnostic markers for the early detection of these pathogens in the field.