Fusarium graminearum, the predominant causal agent of Fusarium head blight (FHB) in cereal crops, impacts the North American wheat industry mainly through the contamination of infected grains with mycotoxins. Understanding the molecular mechanisms underlying adaptive traits in field populations can accelerate the improvement of durably resistant cultivars and effective fungicides. Previously, a Nested Association Mapping Population of F. graminearum (FgNAM) was developed via sexual crosses using Nit-5 (a Nit isolate derived from isolate PH-1) as a recurrent parent and eight other isolates from North America representing phenotypic variation in some adaptive traits, including aggressiveness toward wheat species, DON chemotypes (3ADON vs. 15ADON), and sensitivity to triazole fungicides. This project aims to identify genes associated with these adaptive traits among F. graminearum populations in North America. A pan-genome reflecting diversity in the populations occurring in North America was constructed using nanopore sequencing from the nine isolates as funders for FgNAM. The pan-genome analyses provided additional chromosome-scale reference genomes and annotation based on RNA-seq data obtained from FgNAM founders. Several candidate effectors co-located with hypervariable regions were detected in 3ADON isolate SK1797. Besides, two candidate fungicide insensitivity genes, YC79 (a transcription factor) and MFS1 (a multi-drug transporter) were identified in Metconazole insensitivity isolate 106Eb1 based on fungicide sensitivity phenotyping. This study provides a pan-genome based on the nine FgNAM founders’ genomes combined with transcriptome analysis, which will facilitate the identification of novel genes related to fungicide insensitivity, aggressiveness toward wheat and mycotoxin production in F. graminearum.