Background <p>Tetraploid wheat, comprising both Emmer wheat (<i>Triticum turgidum</i>, AABB genome) and Timopheevii wheat (<i>T. timopheevii</i>, AAGG genome), harbors rich genetic diversity that remains underutilized in modern breeding. The National Bioresource Project-Wheat (NBRP-Wheat) conserves over 2,000 wild and landrace accessions of tetraploid wheat. To enhance the accessibility and application of these resources, we aimed to develop a representative core collection and explore its potential in genetic analysis and breeding.</p> Results <p>We first assessed the genetic diversity of all tetraploid wheat accessions in the NBRP-Wheat gene bank using Diversity Arrays Technology (DArT), and selected 179 representative accessions to establish a core collection. This core set and <i>T. durum</i> cv. Kronos were further genotyped using genotyping by random amplicon sequencing-direct (GRAS-Di). Population structure and diversity analyses revealed clear genetic differentiation among Timopheevii, wild emmer, and domesticated emmer wheat. A significant reduction in genetic diversity was observed in domesticated emmer compared to its wild counterpart. Selective sweep analysis identified several differentiated loci between wild and free-threshing emmer, some of which correspond to known domestication-related genes. Additionally, k-mer-based genome-wide association studies (GWAS) have identified both known and novel loci associated with important agronomic traits, including glume hairiness, anthocyanin pigmentation in cotyledons and coleoptiles, hundred-grain weight, and culm length.</p> Conclusions <p>Our study provides a valuable core collection that captures the genetic diversity of tetraploid wheat conserved in NBRP-Wheat. The identified genomic regions and trait-associated loci demonstrate the utility of this core set for trait dissection and highlight its potential in future wheat improvement programs.</p>