We describe a non-invasive, PCR-RFLP-based method that allows reliable determination of the European water frog species Pelophylax lessonae and Pelophylax ridibundus and the hybrid form Pelophylax esculentus. Maximum-likelihood analysis of ITS2 sequences revealed two robust monophyletic clades corresponding to water frogs of the P. lessonae and P. ridibundus groups. Three restriction enzymes (KpnI, HaeII, and SmaI) were used to digest three conserved ITS2 domains. Taxonomic identification was unambiguous; the three restriction enzymes gave the same results. A French reference sample was identified using allozyme electrophoresis. Our PCR-RFLP method confirmed circa 83% of identification of the allozyme method. We conclude that the difference between identifications was caused by introgression. The western Palearctic water frog (WPWF) group is an impressive example demonstrating the importance of molecular methods for species determination (Hotz 1983; Pagano & Joly 1999; Hotz & Semlitsch 2000; Lodé & Pagano 2000; Plötner & Ohst 2001; Plötner et al. 2001, 2007), for example, in the context of conservation and biodiversity studies. Many WPWF species are morphologically uniform but exhibit specific molecular characters such as unique protein-coding alleles (Plötner & Ohst 2001), microsatellites (Garner et al. 2000; Zeisset & Beebee 2000; Hotz et al. 2001; Christiansen 2005; Holsbeek et al. 2008, 2009a,b), nuclear sequences (Ohst 2008; Plötner et al. 2009), and mitochondrial (mt) DNA markers (Plötner 1998; Holsbeek et al. 2008; Plötner et al. 2008). Widespread hybridization between genetically similar water frog species and lineages (e.g. Akın et al. 2010) may pose serious problems for species determination. In contrast to water frogs from Anatolia and central Asia, the Central and eastern European species Pelophylax lessonae and Pelophylax ridibundus and their hybridogenetic associate Pelophylax esculentus possess distinct morphological and bioacoustic characters that allow an almost exact determination of these forms (Günther 1990; Plötner 2005). Nevertheless, the occurrence of triploid hybrids that possess either two lessonae genomes and one ridibundus genome (LLR) or two ridibundus genomes and one lessonae genome (RRL) hampers identification because of gene-dosage effects (Berger et al. 1986; Vorburger 2001): in LLR individuals, the L genome is expressed twice resulting in a lessonae-like phenotype, while RRL hybrids are often similar to P. ridibundus. In western Europe, the determination of water frogs by morphological and bioacoustic methods may also fail because of extensive hybridization between autochthonous and allochthonous lineages (Günther et al. 1979; Pagano et al. 2003; Christiansen 2005; Holsbeek et al. 2008), which produces intermediate character states for many individuals. Furthermore, character expression depends on age; compared to adults, diagnostic characters are often less clearly expressed in juveniles. Thus, genetic markers are a valuable addition to morphology for identifying adult frogs and even more useful for the determination of juveniles and larvae. Here, we present a relatively inexpensive, rapid, and non-invasive molecular method that allows distinguishing the European water frog forms P. lessonae, P. ridibundus, and P. esculentus with high certainty. Our method is based on specific restriction sites in a 271- bp PCR fragment obtained from the internal transcribed spacer 2 (ITS2) of WPWFs. This ribosomal DNA region has high inter-specific variability and is useful in phylogeny (Van Doninck et al. 2002; Gottschling et al. 2005) and for determination purposes (Joseph et al. 1999; Nikoloudakis et al. 2008), especially in hybrid complexes (Malécot et al. 2009). Ninety-one individuals were collected in Europe and Asia at 28 localities (sample 1, Table 1). Additionally, 93 specimens were collected in southern France at eight localities (sample 2, locality information was given by Pagano et al. 2001a). DNA was isolated from tissue (blood, muscle, and buccal swabs) using QIAmp blood and tissue kits (Qiagen GmbH, Hilden, Germany) or the 5% Chelex 100 ion-exchange resin method (Walsh et al. 1991). Before DNA extraction, ethanol-preserved material was soaked in three changes of distilled water over a 48-h period.