Purpose <p>The generation of stress memory in barley under salt stress conditions is a very important component of plant tolerance and adaptation to adverse environmental conditions. Mechanisms of stress memory allow the plant to "remember" the previous exposure to high salinity and to switch on more quickly pre-established pathways of defense, thus causing less damage and sustaining productivity. Understanding the role of stress memory in barley can thus help inform breeding strategies for salt tolerance cultivar development, which in turn would contribute to stability in crop yield in saline environments using a genome-wide association study (GWAS).</p> Methods <p>To follow the effects of transgenerational, intergenerational, and combined effects of salt stress memory event on the third generation, we compared barley genotypes whose first and second generations were not exposed to salinity stress (C1C2) with the groups that had experienced a single-generation of salt stress either two generations ago (S1C2; first generation salt stress memory effects), or one generation ago (C1S2; second generation salt stress memory effects) and the group that had experienced salinity in both generations (S1S2; combined salt stress memory effects).</p> Results <p>Our results showed that the historical presence of salt stress, regardless of the number of generations previously exposed, had significant effects on changing various agronomic and physiological traits, including spike length, the number of spikelets and grains per spike, grain weight per spike, and thousand kernel weight. More importantly, the history of stress affected both osmolytes and metabolite levels. These results suggest that pretreatment with salt stress could provoke long-lasting changes in barley growth and stress tolerance, which enhance the capacity of its offspring to tolerate the same kind of stress. Based on GWAS analysis, a total of 100 SNP markers located on all chromosomes showed a highly significant association with several potential candidate genes at p-value ≥ 5 with all the measured morphological and biochemical attributes. Interestingly, the expression of our potential genes under salt stress in cereals plays a central role in the plant’s ability to perceive, transduce, and respond to salinity-induced stress signals.</p> Conclusions <p>These genes participate in molecular-mediated pathways that activate critical adaptive responses, such as ion homeostasis, ROS detoxification, and hormonal regulation, which are crucial for plant survival and productivity under saline environments. Knowledge of the function of our potential candidate genes is important for the development of genetic and biotechnological strategies for improving stress tolerance in cereals.</p>