The molecular and physiological work related to cold hardiness in Eucalyptus globulus and the cold-responsive dehydrins is reported. The identification and full-length gene sequence of three dehydrins of 10, 20, and 30 kDa and the comparison of their promoters regarding to potential stress and hormone response elements in E. globulus are shown. The categorization of cold-responsive proteins as dehydrin was based on the similarity in amino acid composition with selected sequenced peptides from chilling-responsive dehydrin reported for other woody plants and the increasing of gene expression level during cold acclimation. The transcript accumulation for these three dehydrin genes increased with cold acclimation and decreased with deacclimation in leaf and stem tissues, being higher in a freezing-resistant genotype of E. globulus compared to a sensitive genotype. By western blot, five dehydrin peptides were identified which increased their expression, under cold stress in leaf and stem tissues. These results provide valuable information about cold acclimation and gene regulation in eucalypt genotypes that differ in their ability to tolerate frost temperature.