Pelophylax perezi is an Iberian green waterfrog with high tolerance to habitat alteration that at times shows local population growth and demographic expansion, even where other species decline. However, pond destruction, invasive predators and hybridisation with other European waterfrog species (P. ridibundus) threaten many of its populations across its range. Hybrids of P. perezi and P. ridibundus (P. kl. grafi) can breed successfully with the former parental species after discarding the whole P. perezi genome in the germinal line, thus representing a sexual parasite for P. perezi. However, little is known about the extent of the contact zone of this hybridogenetic complex. Due to the morphological similarity of the three taxa, molecular tools are needed to delineate their respective ranges. Here we characterise a set of 16 microsatellite markers specifically developed for P. perezi. These markers showed moderate to high polymorphism (2-17 alleles/locus) in two populations from central Spain (n=20 and n=23), allowing individual identification of frogs. Seven of these markers cross-amplified in individuals of P. ridibundus from southern France (3-8 alleles/locus). These markers were used to genotype samples along a transect from southern France to eastern Spain, encompassing both pure and hybrid individuals. Sample assignment to each taxon was based on the new microsatellite loci and compared with nuclear and mitochondrial sequence data. Our results show that these markers are useful to distinguish P. ridibundus, P. perezi and the hybrid form P. kl. grafi from each other, even when sample sizes are low. The newly characterised markers will also be useful in demographic and phylogeographic studies in P. perezi and are thus a valuable tool for evolutionary and conservation oriented research.