Brachypodium distachyon has been highlighted as a model monocot plant with small genome and short life cycle. Biofuels are being developed as renewable energy sources to replace fossil fuels. Bioethanol production is negatively correlated with lignin content. Here, Brachypodium was acutely or chronically irradiated at doses of 50, 100, 150, 200, and 250 Gy. The effect of radiation on plant growth and generation of mutant populations was explored. The lethal effect of radiation was higher in acutely irradiated $M_0$ populations. A dose-dependent negative effect in plant height, tiller number, floral spikelet, and total seed number was observed, with a positive effect in days to heading. The phenotype of 1,773 $M_1$ plants was evaluated, with 417 plants being selected to construct the $M_2$ population. The 31 $M_2$ plants that showed the least staining with phloroglucinol were selected. These mutants could be useful materials for studies such as identification of nucleotide substitutions in genes involved in lignin biosynthesis pathway, monitoring of mutant physiological traits, and evaluation of fitness for bioethanol production. As biological resources, the $M_2$ populations generated in this work will contribute to studies of functional genomics of Brachypodium and to the breeding of grass crops.