Frost tolerance in durum wheat is mainly controlled by copy number variation of CBF - A14 at the Fr - A2 locus. Frost tolerance is a key trait for successful breeding of winter durum wheat (Triticum durum) which can increase the yield performance in regions favoring autumn-sown winter cereals. The aim of this study was to investigate the genetic architecture of frost tolerance in order to provide molecular support for the breeding of winter durum wheat. To this end, a diverse panel of 170 winter and 14 spring durum wheat genotypes of worldwide origin was evaluated for frost tolerance in the field, as well as in a semi-controlled test. A total of 30,611 polymorphic genome-wide markers obtained by a genotyping-by-sequencing approach and markers for candidate loci were used to assess marker-trait associations. One major QTL was detected on chromosome 5A, likely corresponding to Frost Resistance-A2 (Fr-A2). Further analyses strongly support the conclusion that copy number variation of CBF-A14 at the Fr-A2 locus is the causal polymorphism underlying this major QTL. It explains 91.6 % of the genotypic variance and a haploblock of two strongly associated markers in the QTL region also allowed to capture the variance of this QTL. In addition to this major QTL, a much smaller contribution of 4.2 % was observed for Fr-B2. We further investigated this major QTL and found that the copy number of CBF-A14 and the frequency of the frost tolerant haplotype mirrored the climatic conditions in the genotypes' country of origin, suggesting selection through breeding. Two functional KASP markers were developed which facilitate a high-throughput screening of the haploblock and thus a marker-based breeding of frost tolerance in winter durum wheat.