Vernicia montana is a dioecious woody tree with a ZW sex determination system, yet the molecular basis of sex determination remains poorly understood. Here, we constructed a chromosome-level, haplotype-resolved female genome assembly using PacBio HiFi sequencing and Hi-C scaffolding, and integrated population-scale coverage-based genome-wide association analysis of 178 natural individuals to identify sex-associated loci. A highly localized sex-associated signal was detected on ChrB02, defining a ~61.4-kb W-specific sex-determining region (SDR) that is sharply bounded by conserved syntenic blocks but internally enriched in haplotype-specific insertions, duplications, inversions, and a complex inverted-repeat architecture. Polymerase chain reaction assays further validated multiple female-specific segments within this structurally heterogeneous region. Two conserved genes were identified at the SDR boundaries. Notably, VmBASS4.2 displayed pronounced developmental dynamics and broad reproductive expression and is positioned adjacent to major structural rearrangements within the SDR, making it a compelling candidate for functional validation. Heterologous overexpression in Arabidopsis impaired stigma receptivity and increased floral and silique abortion, supporting a dosage-sensitive role in reproductive development. Collectively, our findings suggest that sex determination in V. montana is not driven by the emergence of a novel sex-determining gene but instead is associated with local inverted-repeat-mediated structural remodeling that reshapes the regulatory landscape of pre-existing boundary genes such as VmBASS4.2. This study proposes an inverted-repeat-mediated SDR evolution model, providing a plausible framework linking local structural architecture to regulatory divergence during the early evolution of homomorphic sex chromosomes in plants.

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