Barley is an economically important model for the Triticeae tribe. We recently developed a new resource: the 'Nure' x 'Tremois' mapping population. Two low temperature QTLs were found to segregate on the long arm of chromosome 5H (Fr-H1, distal; Fr-H2, proximal). With the final aim of positional cloning of the genetic determinants of Fr-H1 and Fr-H2, a large segregating population of 1,849 F(2) plants between parents 'Nure' and 'Tremois' was prepared. These two QT loci were first validated by using a set of F(3) families, marker-selected to harbor pairs of reciprocal haplotypes, with one QTL fixed at homozygosity and the alternate one in heterozygous phase. The study was then focused towards the isolation of the determinant of Fr-H2. Subsequent recombinant screens and phenotypic evaluation of F(4) segregants allowed us to estimate (P < or = 0.01) a refined genomic interval of Fr-H2 (4.6 cM). Several barley genes with the CBF transcription factor signature had been already roughly mapped in cluster at Fr-H2, and they represent likely candidate genes underlying this QTL. Using the large segregating population (3,698 gametes) a high-resolution genetic map of the HvCBF gene cluster was then constructed, and after fine mapping, six recombinations between the HvCBFs were observed. It was therefore possible to genetically divide seven HvCBF subclusters in barley, in a region spanning 0.81 cM, with distances among them varying from 0.03 to 0.32 cM. The few recombinants between the different HvCBF subclusters are being marker-selected and taken to homozygosity, to phenotypically separate the effects of the single HvCBF genes.