The nuclear genome sizes of 59 species from 33 genera of the Poaceae subfamily Pooideae were investigated by flow cytometry (FCM). This subfamily is characterized by a wide range of holoploid (2C values) and monoploid (1Cx values) genome sizes and mean chromosome sizes (MC), including both the highest and some of the lowest values of the entire grass family. For example, the tribe Brachypodieae has the smallest monoploid genomes and chromosomes, followed by the majority of Stipeae and individual representatives of the tribes Ampelodesmeae, Duthieeae and Meliceae, which belong to the phylogenetically ‘early-diverging’ lineages. Comparatively large genome and chromosome sizes were found in the Lygeeae and some Meliceae. The ‘core Pooideae’ had the largest values in the subfamily, with the greatest variation in Aveneae, Festuceae and Poeae. The tribes Bromeae and especially Triticeae, which includes wheat and related crops, had larger minimum monoploid genome and chromosome sizes compared to the other ‘core Pooideae’ tribes. It appears that the occurrence of exclusively rather large monoploid genomes (> 3.4 pg/1Cx) and chromosomes (MC ≥ 0.5 pg) is restricted to Triticeae. The origin of x = 7 of the ‘core Pooideae’ from x = 12 of the ‘early-diverging’ Pooideae lineages was apparently not related to an increase in genome size, whereas chromosome fusion caused an increase in chromosome size. The evolutionary aspects of chromosome base number variation in Pooideae are discussed, and new chromosome numbers are presented, including the first polyploid (2n = 4x = 20) of the model plant Brachypodium distachyon s.s.