Abstract To better understand the evolution of virulence we are interested in identifying the genetic basis of this trait in pathogenic fungi and in developing tools for the rapid characterization of variation in virulence among populations associated with epidemics. Fusarium oxysporum f. sp. vasinfectum (FOV) is a haploid fungus that causes devastating outbreaks of Fusarium wilt of cotton wherever it is grown. In the United States, six nominal races and eleven genotypes of FOV have been characterized based on the translation elongation factor (EF-1α) gene and intergenic spacer region (IGS), but it is unclear how race or genotype based on these regions relates to population structure or virulence. We used genotyping-by-sequencing to identify SNPs and determine genetic diversity and population structure among 86 diverse FOV isolates. Six individuals of Fusarium oxysporum closely related to FOV were genotyped and included in some analyses. Between 193 and 354 SNPs were identified and included in the analyses depending on the pipeline and filtering criteria used. Phylogenetic trees, minimum spanning networks (MSNs), principal components analysis (PCA), and discriminant analysis of principal components (DAPC) demonstrated that races and genotypes of FOV are generally not structured by EF-1α genotype, nor are they monophyletic groups with the exception of race 4 isolates, which are distinct. Furthermore, DAPC identified between 11 and 14 genetically distinct clusters of FOV, whereas only eight EF-1α genotypes were represented among isolates; suggesting that FOV, especially isolates within the widely distributed and common race 1 genotype, is more genetically diverse than currently recognized.