Conventional breeding relies on genetic variability in crop germplasm and wild species. This study evaluated 137 barley Recombinant Chromosome Substitution Lines (RCSLs), originating from crossing a drought and salinity- tolerant wild Hordeum spontaneum with the high-quality malting H. vulgare cv. Harrington. The objectives of this work were to i) improve understanding of the genetic diversity and introgression levels in RCSLs using a comprehensive set of high-density single nucleotide polymorphism (SNP) markers; ii) investigate the phenotypic variability of physiological, agronomic, and malting quality traits in RCSLs under rainfed and fully irrigated conditions; iii) unravel the relationship between these traits and the resilience of crops in drought-prone regions; and iv) identify specific SNPs associated with both drought tolerance indices and malting quality traits. Variability in physiological, agronomic, and malting quality traits was examined under rainfed and irrigated conditions, aiming to identify specific SNPs associated with both drought tolerance indices and malting quality traits. Additionally, a subset of three recombinant lines and the parent cv. Harrington was tested in growth chambers under well-watered and water-limited conditions. Agronomic traits were influenced by genotype-by- environment interaction, with notable drought tolerance lines. The grain yield was significantly correlated with yield-related traits and carbon isotope discrimination in grains. Drought stress impacted agronomic and physiological traits, with a strong reduction in leaf gas exchange and chlorophyll and protein content. The H. spontaneum genome introgression into H. vulgare cv. Harrington varied across 137 RCSLs, averaging 13.0 %. Associations between traits and genetic markers were found, particularly for the stress tolerance index in chromosome 1H and for grain quality traits (alfa-amylase, wort color, and protein) in chromosomes 1H, 4H, and 5H. These findings uncovered valuable barley RCSLs that demonstrate drought tolerance, offering promising prospects for breeding programs targeted at improving stress resilience.