<p>Understanding heritable tolerance to concurrent drought and salinity is essential for breeding resilient barley. We combined multi-cycle phenotyping with high-density GWAS across three generations (S1–S3) in a 139-line panel. Antioxidant capacity strengthened progressively: median superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase rose 30–45% in S1 versus controls, increased another 10–15% in S2, and by S3 exceeded controls broadly, whereas unstressed lines drifted less than 5%. Morphological and yield traits showed the opposite pattern: plant height, spike length, and grain number declined about 40% in S1, partially rebounded by 15–25% in S2, and converged on controls in S3, indicating durable trans- and inter-generational acclimation. GWAS resolved twenty-eight SNPs jointly explaining 42% of variance in an antioxidant composite index and 37% of yield stability under combined stress. Two hotspots on chromosomes 4H and 5H harbored pleiotropic loci coupling spike development with redox genes. Markedly, qRT-PCR validation showed that the tolerant genotype mounted stronger transcriptional responses than the susceptible genotype. An early phase (6–12 h) featured rapid induction of calmodulin (<i>HvCaM</i>) and a transient rise in an auxin-responsive gene (<i>HvAUX</i>), consistent with calcium signaling. A sustained phase (12–48 h) showed up-regulation of cell-wall invertase (<i>HvCWIN</i>) and a vacuolar-sorting protein (<i>HvVSP</i>), indicating reinforced carbon partitioning and intracellular trafficking. A late phase (24–48 h) featured activation of anthocyanin synthase (<i>HvANS</i>), consistent with enhanced antioxidant capacity, while α/β-hydrolase (<i>HvABH</i>) declined in the susceptible line. These phased markers prioritize targets for functional analysis and allele stacking to improve tolerance to concurrent drought and salinity in agriculture worldwide.</p>