The cold-regulated (COR14) protein of 14 kDa is a polypeptide accumulated under low-temperature conditions in the chloroplasts of barley leaves. In H. vulgare the COR14 antibody cross-reacts with two proteins, with a slightly different relative molecular weight around the marker of 14.4 kDa, referred to as COR14a and COR14b (high and low relative molecular weight, respectively). In a collection of H. spontaneum genotypes a clear polymorphism was found for the corresponding COR proteins. While some accessions showed the same COR pattern as cultivated barley, in 38 out of 61 accessions examined the COR14 antibody cross-reacted with an additional coldregulated protein with a relative molecular weight of about 24 kDa (COR24). The accumulation of COR24 was often associated with the absence of COR14b; the relationship between the COR14b/COR24 polymorphism and the adaptation of H. spontaneum to different environments is discussed. By studying COR14 accumulation in cultivated barley we have found that the threshold induction-temperature of COR14a is associated with the loci controlling winter hardiness. This association was demonstrated by using either a set of 30 cultivars of different origin, or two sets of frost-tolerant and frost-sensitive F1 doubled-haploid lines derived from the cross Dicktoo (winter type) x Morex (spring type). These results suggest that the threshold induction-temperature of COR14a can be a potential biochemical marker for the identification of superior frostresistant barley genotypes.