BACKGROUND : One of the main goals of the plant breeding in the twenty-first century is the development of crop cultivars that can maintain current yields in unfavorable environments. Landraces that have been grown under varying local conditions include genetic diversity that will be essential to achieve this objective. The Center of Plant Genetic Resources of the Spanish Institute for Agriculture Research maintains a broad collection of wheat landraces. These accessions, which are locally adapted to diverse eco-climatic conditions, represent highly valuable materials for breeding. However, their efficient use requires an exhaustive genetic characterization. The overall aim of this study was to assess the diversity and population structure of a selected set of 380 Spanish landraces and 52 reference varieties of bread and durum wheat by high-throughput genotyping. RESULTS : The DArTseq GBS approach generated 10 K SNPs and 40 K high-quality DArT markers, which were located against the currently available bread and durum wheat reference genomes. The markers with known locations were distributed across all chromosomes with relatively well-balanced genome-wide coverage. The genetic analysis showed that the Spanish wheat landraces were clustered in different groups, thus representing genetic pools providing a range of allelic variation. The subspecies had a major impact on the population structure of the durum wheat landraces, with three distinct clusters that corresponded to subsp. durum, turgidum and dicoccon being identified. The population structure of bread wheat landraces was mainly biased by geographic origin. CONCLUSIONS : The results showed broader genetic diversity in the landraces compared to a reference set that included commercial varieties, and higher divergence between the landraces and the reference set in durum wheat than in bread wheat. The analyses revealed genomic regions whose patterns of variation were markedly different in the landraces and reference varieties, indicating loci that have been under selection during crop improvement, which could help to target breeding efforts. The results obtained from this work will provide a basis for future genome-wide association studies.