Background <p>Japanese oak wilt (JOW), caused by the fungus <i>Dryadomyces quercivorus</i>, has caused mass mortality of healthy oak trees in urban and landscape forests in Japan since the 2000s. The expanding geographic range of the pathogen, vectored by the ambrosia beetle <i>Platypus quercivorus</i>, threatens oak-dominated ecosystems and the timber industry globally. While variation in virulence among isolates has been described based on canker formation within Japanese oak species, there is limited understanding of the genetic variation of <i>D. quercivorus</i> that may underpin this virulence variation. Understanding the genetic variation represents a critical component to determining the capacity of this species to evolve novel virulences or overcome management strategies. In this study, improved hybrid assemblies of the JOW pathogen and closely related species including the Korean oak wilt pathogen (<i>D. quercus-mongolicae</i>) and the Mediterranean oak borer fungal symbiont (<i>D. montetyi</i>) were generated using Oxford Nanopore Technology long reads and Illumina short reads. To understand the genetic variation among JOW fungal populations, 32 isolates from two beetle lineages (A and B) across the Japanese archipelago were sequenced to assess population structure, genetic diversity, and evolutionary potential.</p> Results <p>High-quality genomes were generated for <i>D. quercivorus</i>, <i>D. quercus-mongolicae</i>, and, for the first time, <i>D. montetyi</i>. DAPC, phylogenetic, and distance-based analyses, together with ADMIXTURE patterns at K = 2, revealed two biologically meaningful genetic groupings that broadly corresponded to beetle lineage. Isolates from beetle lineage B, primarily collected in a previous study on the eastern coast of Japan, clustered separately from most isolates associated with beetle lineage A, which were collected across other regions of Japan. Notably, this genetic structure did not correlate with previously reported virulence levels. Window-based Tajima’s D values were generally positive, a pattern consistent with weak bottlenecking and/or structured genetic variation rather than recent population expansion. Furthermore, evidence for sexual recombination was found. Both mating-type idiomorphs were present, and their overall frequency did not significantly deviate from a 1:1 ratio.</p> Conclusions <p>This research shows that the major genetic structure observed in this ambrosia fungus broadly parallels that of its beetle vector lineages. This co-structuring, together with evidence consistent with sexual recombination, suggests that beetle-mediated introductions could transport multiple pathogen genotypes with the potential for recombination. This information is critical for biosecurity risk assessment and will be useful for tracking incursions of this pathogen outside of Japan and for developing effective management strategies, including the use of the genomic resources generated here for rapid diagnostics.</p>