DNA-based techniques are important tools for species assignment, in particular when identification with morphological criteria is difficult. The aim of this study was to genetically determine the species identity of tree frogs (Hyla spp.) populations from western and northern Switzerland (Swiss Plateau), this area being frequently subjected to introductions of species or sub-species from south of the Alps. We sequenced 261 base pairs of the mitochondrial DNA cytochrome b gene from 24 samples of tree frogs from the Swiss Plateau, Ticino (southern Switzerland) and the Dombes region (Ain, France), and compared them with homologous sequences retrieved from DNA databases. The phylogenetic analyses revealed two distinct clades. The first one is represented by samples of Green tree frog (Hyla arborea) from the Swiss Plateau, France, Germany and Greece, confirming the current knowledge about the species' distribution. The second clade includes samples belonging to the Italian tree frog (Hyla intermedia) from south of the Alps (Ticino and Italy), and unexpectedly from the Grangettes site in western Switzerland. These results suggest the introduction of the Italian tree frog H. intermedia north of the Alps, and raise questions about the management of the Grangettes protected area.