To improve our knowledge on the genetic control of drought tolerance, the Oregon Wolfe Barleys (OWB), considered as a reference population in genetic mapping, were subjected to various types of water deficit. Overall, when investigating numerous environments and replications, 40 QTLs were identified in three developmental stages. Based on these loci five QTL clusters were separated, which affect various drought-related traits in at least two developmental stages. Several candidate genes were identified for each QTL cluster using an expressed sequence tag (EST)-based map with high marker density. The putative role of the candidates in drought tolerance is discussed. The phenotypic effect of three of the five candidate genes was also tested on 39 barley landraces and cultivars and a significant relationship was found between the allelic composition of these genes and yield production under stress conditions. This study presents a relevant example of the use of reliable QTL data in the candidate gene approach, while also demonstrating how the results could be practically utilized in marker-assisted selection (MAS).