The model grass species Brachypodium distachyon has three cytotypes that are currently regarded as part of a single species. Catalán et al. combine analysis of phenotypic traits with multiple cytogenetic analyses and detect significant differences between the cytotypes and demonstrate stability of characters in natural populations. Genome size estimations, GISH, FISH and CCP confirm that the 2n = 10 and 2n = 20 cytotypes represent two different diploid taxa, whereas the 2n = 30 cytotype represents a derived allotetraploid from them. They keep the name B. distachyon for the 2n = 10 cytotype, and describe two novel species as B. stacei for the 2n = 20 and B. hybridum for the 2n = 30 cytotypes.