A Potamogeton hybrid found growing in the absence of parental species in a South Bohemian stream, Czech Republic, was subjected to molecular analyses to identify its exact identity. RFLP of the ITS region confirmed its previous morphological identification as P. natans × P. nodosus (= P. ×schreberi). A comparison of its RFLP pattern with those of P. gramineus, P. lucens and P. polygonifolius unambiguously excluded the possibility that the investigated plants are specimens of other similar hybrids (P. ×fluitans, P. ×sparganiifolius, P. ×gessnacensis). The discovery of P. ×schreberi in South Bohemia is the first record of this hybrid for the Czech Republic. So far, it is known only from five countries and the Czech clone is one of a few extant clones of this hybrid in Central Europe. Chloroplast DNA sequencing identified P. nodosus as the maternal parent although at present this species neither occurs at the locality, nor upstream, nor in the entire drainage basin. The other species, P. natans, only occurs downstream of the locality in isolated side pools in a former stream bed and fishponds in an adjacent drainage basin. The available data indicate that this hybrid has persisted vegetatively at this locality for some time in the absence of its parents.