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The development of adaptation strategies for crops under ever-changing climate conditions is a critically important food security issue. Studies of barley responses to ionising radiation showed that this evolutionarily ancient stress factor can be successfully used to identify molecular pathways involved in adaptation to a range of abiotic stressors. In order to identify potential molecular contributors to abiotic stress resilience, we examined the transcriptomic profiles of barley seedlings after exposure to γ-rays, electrons, and protons. A total of 553 unique differentially expressed genes with increased expression and 124 with decreased expression were detected. Among all types of radiation, the highest number of differentially expressed genes was observed in electron-irradiated samples (428 upregulated and 56 downregulated genes). Significant upregulation after exposure to the three types of radiation was shown by a set of ROS-responsive genes, genes involved in DNA repair, cell wall metabolism, auxin biosynthesis and signalling, as well as photosynthesis-related genes. Most of these genes are known to be involved in plant ROS-mediated responses to other abiotic stressors, especially with genotoxic components, such as heavy metals and drought. Ultimately, the modulation of molecular pathways of plant responses to ionising radiation may be a prospective tool for stress tolerance programmes.