Barley (Hordeum vulgare L.), a cornerstone of global cereal crops, is increasingly vulnerable to concurrent heat stress, a critical abiotic factor that is intensified by climate change. This study employed genome-wide association studies (GWAS) to investigate "stress memory," a phenomenon where prior stress exposure enhances a plant's response to subsequent stress events. In this study, we analyzed essential agronomic traits, including plant height, spike length, grain number, and thousand-kernel weight, in conjunction with biochemical markers such as chlorophyll content, proline, and soluble proteins. These assessments spanned three successive generations under heat stress, capturing transgenerational and intergenerational effects and uncovering the cumulative impacts of prolonged stress in the third generation. Markedly, our findings highlight the critical influence of heat stress on plant physiology across generational scales, showcasing significant reductions in chlorophyll content, which reflect stress-induced limitations on photosynthetic capacity. In contrast, the observed consistent and substantial increases in proline and soluble protein content across transgenerational, intergenerational, and third-generation stress memory stages underscore their vital roles in stress mitigation and cellular homeostasis. These results provide compelling evidence of generational stress memory, suggesting potential adaptive strategies that plants employ to cope with harsh environmental conditions. Interestingly, identifying significant SNP markers within key genomic regions using GWAS analysis further highlights the potential for harnessing these loci in breeding programs. These results shed light on the intricate mechanisms of barley's stress tolerance and underscore the potential of integrating genomic, epigenomic, and advanced phenotyping tools into breeding programs to develop heat-resilient cultivars.