The genusNuphar(Nymphaeaceae) comprises aquatic plant species inhabiting freshwater bodies of the Northern hemisphere temperate regions known as yellow water-lilies.Nuphar luteaandN. pumilaare the only representatives in the European continent and present different ecologies: the former is a widespread generalist, while the latter is restricted to northern latitudes or high-altitudes due to its requirements for colder and oligotrophic waters. The Central Europe mountainous areas, the Massif Central (France) and the Cantabrian Mountains (north Iberian Peninsula) harbor relict isolatedN. pumilapopulations endangered by eutrophication and hybridization withN. lutea. We aim to detect hybridization processes in the Massif Central and Cantabrian Mountains populations and compare the genetic diversity ofN. pumilain the relict populations of Central Europe by using microsatellite (SSR) markers. No evidence of hybridization was found in the Iberian population, whereas the admixture betweenN. pumilaandN. luteain the Massif Central populations could be due to hybridization or ancient introgression. Our current knowledge would benefit from genetic diversity studies focusing on both species throughout their distributional range. The Iberian and Massif CentralN. pumilapopulations were genetically distinct, representing two different clusters from other relict populations, with low genetic diversity and a genetic boundary within Central Europe.