In the context of the European Water Framework Directive (2000/60), aquatic macrophytes have become important elements for ascribing the correct ecological value to water systems. Callitriche includes macrophyte species which are widespread and particularly sensitive to environmental cues. High plasticity and morphological variability of these plants have carried to great difficulties in their correct taxonomic determination. Diverse populations of Callitriche of the Po Valley (Northern Italy) have been studied. Plants of Callitriche spp. were collected in Northern Italian rivers with mesotrophic and eutrophic waters and their morphological, karyological, and molecular features were analyzed. Populations with a different chromosome number equal to 10, 18, or 20 were found. Chloroplast rbcL DNA showed the presence of three different sequences. Moreover, the presence of heterozygous rDNA-ITS sequences suggests the existence of a considerable number of hybrids. At ecological level, only one taxon lives in eutrophic waters, while all the others live in mesotrophic ones. Different morphological characters and chromosome set suggest the presence of a new taxon among Italian Callitriche populations. In conclusion, this research underlines that the possibility of Callitriche taxon misclassification, using only the "canonical" macro-morphological approach, is significant and that the incorrect determination of these plants would lead to erroneous evaluation of water quality.