Members of Ranunculus sect. Batrachium are widely distributed and one of the most difficult groups of aquatic plants to identify. Novel DNA-based studies provide an opportunity to elucidate the main evolutionary processes shaping variation in this group and serve as a starting point for further, consistent taxonomic studies of the section. One issue which needs clarification is the role of hybridization in speciation within Ranunculus sect. Batrachium. In this paper we test the molecular character of heterophyllous Ranunculus penicillatus occurring in a few localities in Poland, based on nuclear and plastid DNA sequence data. We found that ITS sequences obtained from this tetraploid taxon displayed a clearly additive pattern between ITS sequences of Ranunculus fluitans and Ranunculus peltatus demonstrating its hybrid origin. The presence of both parental types of matK sequences, even within the same population, shows the multiple origins of the populations studied and suggests that hybridization between R. fluitans and R. peltatus is not rare. In the light of our results we discuss the taxonomic relationships between heterophyllous R. penicillatus and the hybrid R. fluitans × R. peltatus. In addition, based on molecular analysis of 44 individuals of Ranunculus sect. Batrachium representing different morphotypes and originating from different areas, we discuss the resultant genetic groups in the relation to traditionally recognized taxa.