AbstractThe allozyme variation among water frogs of the speciesR. balcanica, R. levantinaandR. ridibunda, all formerly considered as one species (R. ridibundaPallas, 1771), was studied using horizontal starch gel electrophoresis. Blood samples (N # 63) of frogs were collected from five populations in Greece and Israel. Samples (N = 9) of the hybrid frogR. esculentacollected from a locality in Germany were used as an outgroup for phylogenetic analyses. Fifteen enzymes controlled by twenty presumptive loci were identified. Thirteen loci were polymorphic within or among the studied populations. Genetic differentiation among the species was considerably greater than among populations of the same species. Even at Nestos River whereR. ridibundaandR. balcanicaoccur in the same habitats, individuals could be assigned to either species due to characteristic differences of the genotypes (GPI1). This indicates reproductive isolation among these species. The reconstruction of phylogenetic relationships among the three species based on the allozyme data corroborated the model presented on the basis of bioacoustic data:R. ridibundaandR. balcanica(Nei'sgenetic distance D = 0.0820) are sibling species pertaining to an Eurasian lineage, whereasR. levantina(distance to the European species D = 0.1780 ‐ 0.1955) together withR. perezirepresent an independent afroasian lineag