Fusarium wilt of watermelon, caused by
Fusarium oxysporum
f. sp.
niveum
(FON), occurs worldwide and is responsible for substantial yield losses in watermelon-producing areas of the southeastern United States. Management of this disease largely relies on the use of integrated pest management (i.e., fungicides, resistant cultivars, crop rotation, etc.). Knowledge about race structure and genetic diversity of FON in the southeastern US is limited. To determine genetic diversity of the pathogen, FON isolates were collected from symptomatic watermelon plants in commercial fields in Georgia and Florida, USA, and identified based on morphological characteristics and PCR analysis using FON-specific primers. Discriminant analysis of principal components (DAPC) of 99 isolates genotyped with 15 simple sequence repeat (SSR) markers grouped the isolates in eight distinct clusters with two prominent clusters (clusters 1 and 8). Cluster 1 consisted of a total of 14 isolates, out of which 85.7% of the isolates were collected in Florida. However, most of the isolates (92.4%) in cluster 8 were collected in Georgia. Both DAPC and pairwise population differentiation analysis (Ф
PT
) revealed that the genetic groups were closely associated with geographical locations of pathogen collection. Three races of FON (races 0, 2 and 3) were identified in the phenotypic analysis; with race 3 identified for the first time in Georgia. Overall, 5.1%, 38.9% and 55.9% of the isolates were identified as race 0, race 2 and race 3, respectively. The majority of the isolates in cluster 1 and cluster 8 belonged to either race 2 (35.6%) or race 3 (45.8%). Additionally, no relationship between genetic cluster assignment and races of the isolates was observed. The information obtained on genotypic and phenotypic diversity of FON in the southeastern US will help in development of effective disease management programs to combat Fusarium wilt.