Water use efficiency (WUE) is a trait of prime interest in cases of drought stress because it provides information on biomass production in limited water conditions. In order to get information on WUE and additional water use related traits, i.e., dry weight (DW), fresh weight (FW), total leaf water (LW) and leaf water content (WC), greenhouse pot experiments were conducted on 156 barley genotypes (Hordeum vulgare L.) for control (70% maximal water capacity of soil) and drought stress conditions (20% of the maximal water capacity of soil). Significant correlations between WUE and the other water use related traits (r ≤ 0.65) were determined in juvenile barley, and genotypes suited for improving drought stress tolerance in early developmental stages were identified. Furthermore, based on the significant effects of genotypes and treatments, as well as their interaction, data were used for genome wide association studies (GWAS) resulting in the identification of 14 marker trait associations (MTAs) corresponding to four quantitative trait loci (QTL). For WUE, four MTAs were detected mostly located on barley chromosome 4H. For four MTAs, functional annotations related to the involvement in response to abiotic stress were found. These markers may be of special interest for breeding purposes in cases when they will be validated and also detected in later growth stages.