Frost tolerance is the main component of winter hardiness for cereals and is largely dependent on the plant's ability to cold acclimate by means of a slow adaptive response induced by low, non-freezing temperatures. In the present work, a Genome-Wide Association mapping study has been performed, looking for the genetic determinants of frost tolerance in a panel of European spring 2-row barley cultivars. Frost damage was evaluated by measuring chlorophyll fluorescence (F/F) and membrane integrity (Electrolyte Leakage) and considerable variation for this trait was discovered. Genetic association of the trait was sought with >5000 gene-based mapped SNP markers using a Mixed Linear Model approach. A simple genetic architecture was revealed for frost tolerance in this population, with a principal role for the Fr-H1/Vrn-H1 and Fr-H2 loci already detected in previous linkage and association mapping studies. Allelic richness might exist at these loci, not only between winter and spring barley, but also within the spring germplasm, thus offering the opportunity for future development of new spring barley lines with improved resilience in the face of emerging climate change.