Despite their complex evolutionary histories, aquatic plants are highly underrepresented in contemporary biosystematic studies. Of them, the genus Callitriche is particularly interesting because of such evolutionary features as wide variation in chromosome numbers and pollination systems. However, taxonomic difficulties have prevented broader investigation of this genus. In this study we applied flow cytometry to Callitriche for the first time in order to gain an insight into evolutionary processes and genome size differentiation in the genus. Flow cytometry complemented by confirmation of chromosome counts was applied to an extensive dataset of 1077 Callitriche individuals from 495 localities in 11 European countries and the USA. Genome size was determined for 12 taxa. The results suggest that many important processes have interacted in the evolution of the genus, including polyploidization and hybridization. Incongruence between genome size and ploidy level, intraspecific variation in genome size, formation of autotriploid and hybridization between species with different pollination systems were also detected. Hybridization takes place particularly in the diploid-tetraploid complex C. cophocarpa-C. platycarpa, for which the triploid hybrids were frequently recorded in the area of co-occurrence of its parents. A hitherto unknown hybrid (probably C. hamulata × C. cophocarpa) with a unique chromosome number was discovered in the Czech Republic. However, hybridization occurs very rarely among most of the studied species. The main ecological preferences were also compared among the taxa collected. Although Callitriche taxa often grow in mixed populations, the ecological preferences of individual species are distinctly different in some cases. Anyway, flow cytometry is a very efficient method for taxonomic delimitation, determination and investigation of Callitriche species, and is even able to distinguish homoploid taxa and identify introduced species.