The first attempt to artificially hybridize species of Potamogeton resulted in the hybrid P. perfoliatus × P. gramineus. The morphological features, reproductive behaviour and molecular markers of the offspring of this experimental hybridization were compared with those of the parental species and natural hybrids of the same assumed parentage. A phenotype corresponding to that of the natural hybrid P. × nitens was acquired from an experimental cross between P. perfoliatus and P. gramineus. All plants, both natural and artificial, of this hybrid were consistently sterile. They showed the ITS variants of both parental taxa, which is consistent with biparental inheritance of nuclear DNA. The experimental hybrid was used to test the maternal inheritance of chloroplast DNA in Potamogeton. Sequences of a chloroplast intergenic spacer (rpl20- rps12) were identical with those of the female parent. Then, the directions of the crosses resulting in the natural hybrids were investigated. Of five natural populations of P. × nitens, P. gramineus was the maternal parent of two and P. perfoliatus of three populations. The frequency of hybridization events and rise of hybrids are discussed.