Drought is a major threat to world agriculture. In order to identify proteins associated with plant drought tolerance, barley varieties bred in the UK (Golden Promise) and Iraq (Basrah) were compared. The variety Basrah showed physiological adaptations to drought when compared to Golden Promise, for example relative water content of roots and shoots after 1 week of drought was much higher for Basrah than for Golden Promise. DIGE analysis was carried out on proteins from roots and leaves under control and drought conditions. Twenty-four leaf and 45 root proteins were identified by MALDI-TOF MS. The relative expression patterns of the identified proteins fell into a number of distinct classes. The variety Basrah is characterised by constitutive expression or higher drought-induced expression levels of proteins regulating ROS production and protein folding. Photosynthetic enzymes, by contrast, were downregulated in Basrah. Enzyme assays showed a good correlation between DIGE-derived protein abundance estimates and enzyme activity in extracts. Overall, this study shows that the enhanced drought tolerance of variety Basrah is driven by an enhanced regulation of ROS under drought.