Over 125 year ago, Fusarium wilt of cotton was first described on infected plants from fields in Alabama. Since then the causative agent, Fusarium oxysporum f. sp. vasinfectum (Fov), has been isolated from all major cotton production areas worldwide. Despite the prevalence of this pathogen, little in known on a molecular level how the fungus is able to cause disease. In an effort to understand the virulence factors required to colonize cotton, the genomes of five Fov isolates were sequenced including a representative of the highly virulent race 4 genotype. Comparative genomic analysis between these isolates and other members of the F. oxysporum species complex revealed that large portions of the Fov lineage-specific chromosomes share syntenic regions. Interestingly, the genome of the race 4 isolate was significantly larger than the other Fov genomes and ~25% of the genome was identified as lineage-specific. Several genes of interest that could be involved in pathogenicity on cotton are currently being investigated, including putative small secreted proteins that may serve as effectors and secondary metabolites. One example is a copper specific extracellular superoxide dismutase encoded by FoSOD5 which is induced by plant carbon sources, confers tolerance to reactive oxygen species, and is required for full virulence on cotton. To aid in evaluation of potential Fov virulence factors for cotton, we have developed a CRISPR/Cas9 ribonucleoprotein mediated transformation system that can be used to generate mutants and facilitate gene editing.