Isolates of the tomato wilt pathogen Fusarium oxysporum f. sp. lycopersici, predominantly from commercial tomato fields in Florida and southwestern Georgia, were characterized using vegetative compatibility grouping (VCG), nuclear restriction fragment length polymorphism (RFLP), and virulence. All field isolates that could be grouped into VCG belonged to VCG 0033. This VCG was first described by Marlatt et al. in 1996 for isolates from northern Florida, Arkansas, and North Carolina. This study demonstrates that VCG 0033 is also widespread in central and southern Florida, in addition to southwestern Georgia, and also was found to be present in Puerto Rico. Population genetic and phylogenetic analyses of 121 isolates indicated that molecular diversity among VCG 0033 isolates was by far the highest in Manatee County, FL, suggesting it to be the probable center of origin of this relatively newly described VCG. Virulence tests with a subset of isolates identified all VCG 0033 isolates as race 3, although differences in aggressiveness were observed among tested isolates, independent of resistance genes in the differential cultivars. The widespread VCG 0030 of F. oxysporum f. sp. lycopersici was not present in our field collections. This was unexpected, as strains from Florida isolated prior to 1990 were predominantly VCG 0030. This would suggest that VCG 0033 has replaced VCG 0030 in recent years in commercial tomato fields of Florida and southwestern Georgia.