The European water frog (Pelophylax esculentus) complex represents a unique and adequate model system for the study of interspecific hybridization and the mechanisms enabling interspecific hybrids to overcome the reproductive barriers. The difficulties in the study of individuals from the P. esculentus complex are associated with high polymorphism of morphological characters in parental species and interspecific hybrids, as well as with the presence of polyploid hybrid forms. From the discovery of the phenomenon of interspecific hybridization and the demonstration of successful reproduction of interspecific hybrids, researchers constantly searched for the methods necessary for the most accurate identification of parental species and various hybrid forms. This review describes biochemical, cytogenetic, and molecular methods and approaches used to identify individuals from the European water frog complex, as well as to analyze the genomes transferred with the gametes of hybrids. The advantages and disadvantages of these approaches are discussed. The presented methods can be used for studying other hybrid complexes of fish, amphibians, and reptiles.