Durum wheat (Triticum durum) is predominantly grown as spring type and depending on the production area autumn or spring sowing is used. For the durum production in Austria and Germany, autumn sowing has several advantages, such as yield increase and stability, but this requires the selection for winter hardiness including a good frost tolerance. The aim of this study was to support breeding of winter durum and to facilitate genomic approaches by molecularly characterizing a panel of 170 diverse winter and 14 spring durum lines employing a genotyping-by-sequencing approach. We obtained an unprecedentedly high number of 30,611 polymorphic markers covering the entire genome. The principal coordinate analysis and the cluster analysis revealed the absence of a major population structure but a tendency of lines to group according to their country of origin. Linkage disequilibrium was found to decay within a short distance of approximately 2–5 cM and also showed variable patterns along chromosomes. In summary, our results can assist breeding of durum wheat and pave the way for genomic approaches towards knowledge-based winter durum breeding.