Abstract Wheat genetic resources hold the diversity required to mitigate agricultural challenges from climate change and reduced inputs. Using DArTseq, we genotype 461 wheat landraces and cultivars and evaluate them for powdery mildew resistance. By developing a k-mer-based GWAS approach with fully assembled genomes of Triticum aestivum and its progenitors, we uncover 25% more resistance-associated k-mers than single-reference methods, outperforming SNP-based GWAS in both loci detection and mapping precision. In total, we detect 34 powdery mildew resistance loci, including 27 potentially novel regions. Our approach underscores the importance of integrating multiple reference genomes to unlock the potential of wheat germplasm.