Fusarium graminearum species complex (FGSC) members cause Fusarium head blight (FHB) of wheat (Triticum aestivum L.) and small grains in the United States. The U.S. population is diverse and includes several genetically distinct local emergent subpopulations, some more aggressive and toxigenic than the majority population. Kentucky is a transition zone between the Mid-Atlantic and Midwestern wheat production areas. Sixty-eight Fusarium strains were isolated from symptomatic wheat heads from central and western Kentucky and southern Indiana in 2007. A multilocus genotyping assay and a variety of additional molecular markers, including some novel markers developed using the F. graminearum genome sequence, were used to characterize the pathogen population. Five of the isolates were identified as members of two non-FGSC species, F. acuminatum and F. cf. reticulatum, but they did not cause symptoms in greenhouse tests. All the FGSC isolates belonged to the 15-ADON chemotype of F. graminearum. Comparative genetic analysis using variable nuclear tandem repeat (VNTR) markers indicated that the population in Kentucky and Indiana belonged to the dominant North American population, with some diversification likely due to local evolution. Telomere and RFLP fingerprinting markers based on repetitive sequences revealed a high degree of genetic diversity within the population, with unique genotypes found at each location, and multiple genotypes isolated from the same head.