Abstract Background Barley (Hordeum vulgare L.) is the fourth most important cereal crop worldwide. Barley production is compromised by many abiotic stresses including drought. Wild barley is a valuable source of alleles that can improve adaptation of cultivated barley to drought stress. Results In the present study, a nested association mapping population named HEB-25, consisting of 1420 BC1S3 lines that were developed by crossing 25 different wild barley accessions to the elite barley cultivar ‘Barke’, was evaluated under both control and drought-stressed conditions in the Australian Plant Phenomics Facility, University of Adelaide. Overall, 14 traits reflecting the performance of individual plants in each treatment were calculated from non-destructive imaging over time and destructive end-of-experiment measurements. For each trait, best linear unbiased estimators (BLUEs) were calculated and used for genome-wide association study (GWAS) analysis. Among the quantitative trait loci (QTL) identified for the 14 traits, many co-localise with known inflorescence and developmental genes. We identified a QTL on chromosome 4H where, under drought and control conditions, wild barley alleles increased biomass by 10 and 17% respectively compared to the Barke allele. Conclusions Across all traits, QTL which increased phenotypic values were identified, providing a wider range of genetic diversity for the improvement of drought tolerance in barley.