Nuclear genome sizes of 54 representative species from 44 genera of the monocot order Poales were investigated by flow cytometry (FCM). Small holoploid genomes with < 2 pg/2C are characteristic of the Poales, only some families have larger 2C values, although this is not consistently the case. The sizes of monoploid genomes as well as mean DNA content per chromosome (MC) show a similar pattern. A comparison of the genome size data with current molecular phylogenetic data suggests that small monoploid genomes (1Cx < 0.4 pg) and small chromosomes (MC ≤ 0.05 pg), as found in some families, are likely the ancestral features of the order Poales. Conspicuous increases in genome size occurred particularly in the Poaceae (grasses) and to a lesser extent in the xyrid clade and the restios. According to previous phylogenomic studies, the Poaceae are characterized by a whole-genome duplication (WGDs) called ρ, which is absent in all other Poales families. However, it is clear from the 1Cx values, that the ρ event is not, or no longer, associated with a significant increase in the minimum 1Cx genome sizes of grasses compared to other Poales families. Future studies need to clarify whether the smallest 1Cx values in the Poaceae are due to a secondary reduction of the nuclear genome after the ρ event and whether the relatively large minimal 1Cx values of the xyrid clade were caused by a further WGD within Poales.