Genetic variation at 28 protein loci was analyzed electrophoretically in populations of the Central European treefrog, Hyla arborea arborea, from Italy, Switzerland, Germany and the Balkans. Two distinct population groups were detected, differing at 9 more or less diagnostic loci, one ranging from Sicily to peninsular Italy, northwards to the Italian slope of the Alps (Italian treefrog), the second parapatrically distributed with respect to the first, north of the Alps, east of the Isonzo river, and southwards to the Balkans (H. arborea arborea s.s.). In the area of close contiguity between the two population groups (approximately coinciding with the border between Italy and Slovenia), neither mixed populations nor hybrid individuals were found. However, a number of specimens with introgressed genotypes were detected in both forms in the contact area and its surroundings, possibly as the result of past hybridization events. The lack of present gene exchange in the contact area indicates that the Italian treefrog and H. arborea arborea (s.s.) are distinct biological species. Average Nei's genetic distance between the Italian treefrog and H. arborea arborea (s.s.) is 0.47, corresponding to an estimated time for evolutionary divergence of about 2-3 million years. The Italian treefrog and H. arborea arborea (s.s.) were found to be morphologically very similar, with a single significant difference (tympanum size) among the considered characters; this is larger in the Italian treefrog. The name Hyla italica nomen novum is proposed for the Italian treefrog, as the name Hylaria variegata assigned by Rafinesque (1814) to treefrog specimens from Sicily is unavailable.