<p>This study provides valuable insights into the genetic diversity and population structure of olive tree (Olea europaea L. subsp europaea) accessions from the National Olive Genetic Resources Collection, Boughrara (Tunisia) (NOGRB) by using genotyping-by-sequencing (GBS).The olive tree is among the oldest and most widely cultivated fruit species grown in the Mediterranean Basin. The National Olive Genetic Resources Collection (NOGRB), established in 1992 in Boughrara, (Sfax, Tunisia), serves as a vital repository for olive genetic diversity.In this study, we examined the genetic diversity and population structure of 90 olive tree genotypes, including 55 cultivars from Tunisia and 35 from other Mediterranean countries. Using a genotyping-by-sequencing (GBS) method, we identified 2,077 single nucleotide polymorphisms (SNPs). Further analyses showed moderate genetic diversity, with an expected heterozygosity (He) of 0.018, observed heterozygosity (Ho) of 0.049, and a low polymorphism information content (PIC) of 0.016, suggesting limited informativeness of the marker. Multivariate analyses were used to assess the distribution of genetic variation within the collection. Bayesian model-based clustering indicated a main grouping at K = 3. Principal component analysis (PCA) and a dendrogram revealed the genetic structure, identifying distinct groups and subgroups among the 90 accessions. These results provide important genomic resources for phylogenetic and population structure studies, supporting olive breeding efforts and the development of the olive oil industry in Tunisia.</p>