The genetic differentiation among 33 populations of the Italian treefrog, Hyla intermedia (Anura: Hylidae), was investigated using both biparentally (23 allozyme loci) and maternally (partial mitochondrial cytochrome b gene) inherited markers. Two main population groups were evidenced by both markers, located north and south of the northern Apennines. However, the pattern of differentiation between these two groups was much less pronounced at allozymes than at mtDNA, leading to gene flow estimates that were 25 times lower at mitochondrial than at nuclear level. Also, the mtDNA divergence between the two groups was particularly marked for two cospecific lineages of anuran amphibians (the P-distance being on average 9.04%), while their average genetic distance at allozymes was comparatively low (D (NEI) = 0.07). This contrasting pattern of nuclear versus mitochondrial genetic variation is discussed in the context of: (1) marker specific selection, (2) secondary contact and sex-biased gene flow and (3) ancestral polymorphism and colonization from north to south. Finally we emphasize how, for population genetic studies, the use of multiple markers having distinct evolutionary properties can help unravel the existence of more complex evolutionary histories.