Dehydrin gene (Dhn) expression is associated with plant response to dehydration. The aim of the present study was to investigate the association of differential expression of Dhn genes (Dhn 1, 3, 5, 6, and 9) with drought tolerance found in wild barley (Hordeum spontaneum). Tolerant and sensitive genotypes were identified from Israeli (Tabigha microsite) and Jordanian (Jarash and Waddi Hassa) populations (based on scoring of water loss rate of 390 genotypes). The five Dhn genes were up-regulated by dehydration in resistant and sensitive wild barley genotypes. Notably, differences between resistant and sensitive genotypes were detected, mainly in the expression of Dhn1 and Dhn6 genes, depending on the duration of dehydration stress. Dhn1 tended to react earlier (after 3 h) and higher (12 h and 24 h) in resistant compared to sensitive genotypes. The level of expression of Dhn6 was significantly higher in the resistant genotypes at the earlier stages after stress. However, after 12 and 24 h Dhn6 expression was relatively higher in sensitive genotypes. The present results may indicate that these genes have some functional role in the dehydration tolerance in wild barley. The authors suggest that the observed differences of Dhn expression in wild barley, originating from different micro- and macro ecogeographic locations, may be the result of adaptive edaphic and climatic selective pressures.