Wintering ability is an important trait in barley, particularly in northern Japan. Although fructan is known to be related to wintering ability in wheat and temperate forage grasses, its contribution to wintering ability in barley remains unclear. To clarify the role of fructan with respect to wintering ability in barley, we analyzed the relationships among wintering ability, shoot fructan content, and the allelic variation of five fructan metabolic enzyme genes, 1-FEH, 6-FEH, 1-FFT, 1-SST, and 6-SFT, in 105 Japanese barley cultivars and breeding lines. The cultivars and breeding lines developed in the colder northern region of Japan exhibited a superior wintering ability and greater fructan content accumulated prior to snow cover than those bred in the warmer southern region. In our sequence analyses of the five fructan metabolic enzyme genes, 6 to 10 genotypes were found in each of the genes in the tested barley cultivars and lines. Cultivars and lines with the same combination of fructan metabolic enzyme gene genotypes were grouped, and the average wintering ability and fructan content values of the different groups were compared. A significant correlation was detected between fructan content and wintering ability, specifically, groups with a higher fructan content prior to snow cover, especially in stem tissue, exhibited a superior wintering ability. The group with the highest wintering ability possessed a unique 6-SFT genotype. The cultivars and breeding lines were classified into three groups with a high, medium, and low wintering ability based on the 1-FEH genotypes. We developed derived cleaved amplified polymorphic sequence (dCAPS) markers that identify the 6-SFT and 1-FEH genotypes observed in the group with the highest wintering ability and in the groups with a high, medium, and low wintering ability, respectively.