Fusarium oxysporum f. sp. cepae causes Fusarium basal rot of onion, a disease of worldwide importance. Limited information is available on the phylogenetic diversity, vegetative compatibility groups (VCGs), mating type idiomorphs, and virulence of F. oxysporum isolates associated with onion. Therefore, these characteristics were investigated in 19 F. oxysporum f. sp. cepae isolates from Colorado, 27 F. oxysporum f. sp. cepae and 33 F. oxysporum isolates nonpathogenic to onion from South Africa. Six F. oxysporum f. sp. cepae VCGs (0421 to 0426) were identified, of which three were new. The dominant VCGs in Colorado and South Africa were VCG 0421 (47% of isolates) and VCG 0425 (74%), respectively. VCG 0423 was the only VCG that was shared between the two regions. Molecular phylogenies (intergenic spacer region of the rDNA, elongation factor 1α, and mitochondrial small-subunit) confirmed the polyphyletic nature of F. oxysporum f. sp. cepae and showed that some F. oxysporum f. sp. cepae and nonpathogenic F. oxysporum isolates were genetically related. Most F. oxysporum f. sp. cepae isolates clustered into two distinct, well-supported clades. The largest clade only contained highly virulent isolates, including the two main VCGs (0421 and 0425), whereas the basal clade mostly contained moderately virulent isolates. These groupings along with the VCG data provide an important basis for selection of isolates for use in breeding programs, and for the development of molecular makers to identify VCGs. Mating type genotyping revealed the distribution of both mating type (MAT1-1 and MAT1-2) idiomorphs across phylogenetic clades, and the fact that several isolates contained both idiomorphs.