DAPI and propidium iodide flow cytometry were used to determine the variation in genome size in 166 samples and of all taxa and ploidy levels of Fallopia section Reynoutria (knotweeds) recorded in the Czech Republic. Significant differences were detected in the amount of nuclear DNA, associated with the ploidy levels and taxonomic identity of the material. At each ploidy level, F. sachalinensis showed the lowest and F. japonica the highest fluorescence intensities. The fluorescence values for the hybridogenous F. ×bohemica were located in-between these two levels. In most cases, there was at least a four-percent gap in fluorescence values between the nearest neighbours belonging to a different taxon. Intraspecific variation in genome size was very low in all taxa except hexaploid F. ×bohemica; this could be due to the complex evolutionary history of this taxon. Our results indicate that the amount of nuclear DNA can be used as a reliable marker for the identification of homoploid knotweed species and their hybrids. Different evolutionary pathways for the origin of high polyploids and/or hybridogenous taxa are proposed based on genome size.