Fusarium head blight (FHB), caused by Fusarium graminearum, is a major disease of wheat worldwide. Regular monitoring of the local pathogen population is critical for understanding pathogen dynamics and developing effective management strategies. In this study, we conducted surveys and collected FHB-symptomatic spikes from wheat fields across eastern North Dakota and western Minnesota during the growth seasons of 2021 and 2022. A total of 300 F. graminearum pure cultures were isolated and used to assess chemotype diversity, aggressiveness and DON production. Chemotyping indicated that 69.40% and 30.58% of the isolates were 15ADON type and 3ADON type, respectively. NX-2 chemotype was only found for one single isolate collected from a wheat field located in Norman County, Minnesota. Aggressiveness and DON production were evaluated on two wheat genotypes, Alsen (moderately resistant to FHB) and Wheaton (highly susceptible to FHB). No significant difference was observed in aggressiveness between 15ADON and 3ADON isolates, although the latter produced significantly higher DON. Genome sequencing and single nucleotide polymorphism (SNP) analysis classified the isolates into three genetic clusters, with two corresponding to the two different chemotypes (15ADON and 3ADON), respectively, and one admixture of the populations. Furthermore, the genome wide association study identified multiple SNPs associated with aggressiveness and DON production on chromosomes 1, 2 and 4, some of which are potential novel candidate genes associated with aggressiveness. Overall, our work provides a new information on current F. graminearum populations in this region, which is important for the development and deployment of resistant cultivars.