Genetic characterization enhances the development of rational conservation strategies and the utilization of germplasm to plant breeding programs. In the present study, 19 microsatellite markers were employed to evaluate the genetic diversity and the genetic affiliations across 20 Cypriot durum wheat (Triticum turgidum L. subsp. durum) landraces, 13 landraces from the broader Mediterranean basin and 22 modern varieties. Cluster analysis depicted a clear separation among modern varieties and landraces, regardless of their origin. Landraces presented the highest genetic variation (average discriminating power of 0.89) and a high number of private alleles (131) was detected; underlying the unique genetic mark-up of this genepool. AMOVA revealed that the highest variability was detected within the landraces originating from Cyprus and landraces from the broader Mediterranean basin. The Cypriot landrace 'Kyperounda' was selected for further evaluation of its' intra-genetic variation and it was determined that genetic diversity was higher in accessions conserved as sublines (He 0.643-0.731) than bulks (He 0.384-0.469). Bayesian analysis revealed substantial admixture within 'Kyperounda' accessions, depicted also by Principal Coordinate Analysis. The findings of the current manuscript emphasize that high intra-genetic diversity is retained when landraces are conserved as sublines in ex situ collections, while landraces that are conserved as bulks have a higher risk of bottleneck. Hence, a more exhausting diversity evaluation is needed in order to fully utilize landraces in breeding schemes and to prevent the loss of genetic variation.