Water scarcity is a critical threat to global crop production. Here, we used the natural diversity of barley (Hordeum vulgare) to dissect the genetic control of proline (Pro) mediated drought stress adaptation. Genetic mapping and positional cloning of a major drought-inducible quantitative trait locus (QPro.S42-1H) revealed unique allelic variation in pyrroline-5-carboxylate synthase (P5cs1) between the cultivated cultivar Scarlett (ssp. vulgare) and the wild barley accession ISR42-8 (ssp. spontaneum). The putative causative mutations were located in the promoter of P5cs1 across the DNA binding motifs for abscisic acid-responsive element binding transcription factors. Introgression line (IL) S42IL-143 carrying the wild allele of P5cs1 showed significant up-regulation of P5cs1 expression compared to Scarlett, which was consistent with variation in Pro accumulation under drought. Next, we transiently expressed promoter::reporter constructs of ISR42-8 and Scarlett alleles in Arabidopsis (Arabidopsis thaliana) mesophyll protoplasts. GUS expression analysis showed a significantly higher activation of the ISR42-8 promoter compared to Scarlett upon abscisic acid treatment. Notably, the ISR42-8 promoter activity was impaired in protoplasts isolated from the loss-of-function abf1abf2abf3abf4 quadruple mutant. A series of phenotypic evaluations demonstrated that S42IL-143 maintained leaf water content and photosynthetic activity longer than Scarlett under drought. These findings suggest that the ancestral variant of P5cs1 has the potential for drought tolerance and understanding drought physiology of barley and related crops.