Brachypodium distachyon (Brachypodium) is now intensively utilized as a model grass species in various biological studies. Its favorable cytological features create a unique foundation for a convenient system in mutagenesis, thereby potentially enabling the 'hot spots' and 'cold spots' of DNA damage in its genome to be analyzed. The aim of this study was to analyze the involvement of 5S rDNA, 25S rDNA, the Arabidopsis-type (TTTAGGG)n telomeric sequence and the Brachypodium-originated centromeric BAC clone CB33J12 in the micronuclei formation in Brachypodium root tip cells that were subjected to the chemical clastogenic agent maleic hydrazide (MH). To the best of our knowledge, this is the first use of a multicolor fluorescence in situ hybridization (mFISH) with four different DNA probes being used simultaneously to study plant mutagenesis. A quantitative analysis allowed ten types of micronuclei, which were characterized by the presence or absence of specific FISH signal(s), to be distinguished, thus enabling some specific rules governing the composition of the MH-induced micronuclei with the majority of them originating from the terminal regions of chromosomes, to be identified. The application of rDNA sequences as probes showed that 5S rDNA-bearing chromosomes are involved in micronuclei formation more frequently than the 25S rDNA-bearing chromosomes. These findings demonstrate the promising potential of Brachypodium to be a useful model organism to analyze the effects of various genotoxic agents on the plant nuclear genome stability, especially when the complex FISH-based and chromosome-specific approaches such as chromosome barcoding and chromosome painting will be applied in future studies.