We used pathogenicity and random amplified polymorphic DNA (RAPD) markers to assess genetic diversity among 46 isolates of Fusarium oxysporum f. sp. vasinfectum of worldwide origin. Based on pathogenicity tests on five differential cotton cultivars and species, isolates were differentiated into three races (A, 3, and 4), restricted to defined geographic areas. The amount of genetic variation was evaluated by polymerase chain reaction amplification with a set of 11 random 10-mer primers. All amplifications revealed scorable polymorphisms among the isolates, and a total of 83 band positions was scored (1/0) for the 11 primers tested. Genetic distances between each of the isolates were calculated, and cluster analysis was used to generate a dendrogram showing relationships between them. Isolates clustered into three groups corresponding to their pathological reactions. We suggest that RAPD markers can be a quick and reliable alternative for differentiating isolates of F. o. vasinfectum into their respective pathogenicity group.