The genus Brachypodium contains about 15 grass species with the most important representative being the model plant B. distachyon. Brachypodium species are frequently polyploid, with small genomes and variable chromosome base numbers of 5, 7, 8, 9, 10. This diversity prompts the use of the genus as a model system for studying karyotype evolution and divergence in grasses. B. distachyon BAC libraries and genome sequence data facilitate detailed comparison of chromosomal organization across the Brachypodium species based on modern molecular cytogenetic techniques, such as BAC-FISH and chromosome painting. Single-locus BACs can be used as molecular cytogenetic markers which are very important for plant genome analysis, especially in species lacking reliable methods for karyotyping. Chromosome painting (CP) is one of the most powerful and spectacular tools of modern molecular cytogenetics, enabling complex analyses of nuclear genome structure and evolution. Here we demonstrate comparative mapping of single BACs and CP with BAC pools representative of individual B. distachyon chromosomes. We analysed genome evolution at the chromosomal level in B. sylvaticum, B. pinnatum and other Brachypodium species. The authors acknowledge financial support from Polish Ministry of Science and Higher Education (grant N N303 570738; N N303 569538). AB, NB and EB acknowledge scholarships from the UPGOW project co-financed by the European Social Fund.