<p>Cold stress is a major constraint on barley productivity, and repeated cold exposure can trigger acclimation responses that persist across generations, indicating a heritable “cold memory” that can be exploited in breeding. We evaluated morpho-physiological and yield responses across three successive generations in a diverse collection of 138 barley genotypes under recurring cold, quantified stress tolerance indices (STIs), profiled trait interrelationships, mapped genome-wide associations, and validated candidate gene expression in contrasting genotypes. First-generation losses in chlorophyll, plant height, spike length, and yield components (≈40–65%) decreased to ~ 15–25% by the third generation, while protein remained elevated, evidence of transgenerational acclimation. Stress tolerance indices (STIs) increased significantly, and trait correlations shifted toward positive coupling between physiology (chlorophyll, proline, and soluble protein) and yield. A genome-wide association study (GWAS) resolved 73 marker–trait association (MTAs) with 67 SNPs across 1H–7H, highlighting stable loci on 1H, 2H, 3H, and 4H implicating ion homeostasis, osmolyte/cell-wall remodeling, MYB/kinase signaling, ROS scavenging, and ubiquitin-mediated turnover. Expression assays corroborated the mechanism: the tolerant genotype mounted a rapid 6–12 h induction of kinase, MYB signaling, antioxidant/peroxidase, cell-wall glycoprotein, β-glucosidase, glutathione transport, and F-box turnover, whereas the susceptible line responded later and weaker. Collectively, the data demonstrate transgenerational, redox-centered acclimation that stabilizes photosynthesis and reproduction, nominate compact genetic hubs for fine-mapping, and provide immediate targets to integrate stress-memory loci into marker-assisted and genomic selection for durable cold-resilient barley.</p> Graphical Abstract <p>A graphical abstract for genome-wide discovery of barley cold tolerance loci coordinating morpho-physiological responses and redox defense networks. A three-generation barley panel (G1–G3) was phenotyped under control and cold-stress conditions. Morpho-physiological and redox/antioxidant traits were quantified and summarized as stress indices (STI). Trait inter-relationships were examined using correlation analyses to define coordinated response modules. Genome-wide association mapping (GWAS) was then conducted using STI-derived phenotypes to identify significant marker–trait associations and pleiotropic loci. Candidate genes were prioritized from associated intervals based on functional annotation and pathway relevance (cold signaling, ROS/redox homeostasis, membrane protection, and growth regulation). Key candidate genes were subsequently validated by qRT-PCR across contrasting genotypes and treatments to confirm cold-responsive expression patterns</p> <p></p>