DNA damage response (DDR), a complex network of cellular pathways that cooperate to sense and repair DNA lesions, is regulated by several mechanisms, including microRNAs. As small, single-stranded RNA molecules, miRNAs post-transcriptionally regulate their target genes by mRNA cleavage or translation inhibition. Knowledge regarding miRNAs influence on DDR-associated genes is still scanty in plants. In this work, an in silico analysis was performed to identify putative miRNAs that could target DDR sensors, signal transducers and effector genes in wheat. Selected putative miRNA-gene pairs were tested in an experimental system where seeds from two wheat mutant lines were irradiated with 50 Gy and 300 Gy gamma(γ)-rays. To evaluate the effect of the treatments on wheat germination, phenotypic and molecular (DNA damage, ROS accumulation, gene/miRNA expression profile) analyses have been carried out. The results showed that in dry seeds ROS accumulated immediately after irradiation and decayed soon after while the negative impact on seedling growth was supported by enhanced accumulation of DNA damage. When a qRT-PCR analysis was performed, the selected miRNAs and DDR-related genes were differentially modulated by the γ-rays treatments in a dose-, time- and genotype-dependent manner. A significant negative correlation was observed between the expression of tae-miR5086 and the RAD50 gene, involved in double-strand break sensing and homologous recombination repair, one of the main processes that repairs DNA breaks induced by γ-rays. The results hereby reported can be relevant for wheat breeding programs and screening of the radiation response and tolerance of novel wheat varieties. •Knowledge of miRNA regulation on genes involved in the DNA Damage Response (DDR) pathway is still limited in plants.•Several miRNAs and DDR-related genes are differentially modulated by γ-rays during wheat germination in a time- and genotype-dependent manner.•An indirect validation of miRNA-target gene relation is provided for the tae-miR5086-RAD50 pair.•The gathered results are relevant for future wheat breeding programs and screening of new varieties with improved radiation response.