Current studies indicate that both processes and mechanisms of natural hybridization go far beyond the formation and maintenance of hybrid zones between species. These studies demonstrate that the evolutionary consequences of hybridization can include extinction or extirpation of lineages but may also favor the formation of new hybrid species in an ecological context. The unambiguous identification of occurrences of hybridization in natural populations is a crucial first step in addressing questions related to natural hybridization in both evolutionary and ecological terms. Here, we provide the first molecular evidence of extensive natural hybridization between two ancient sister species of spectacled salamanders -Salamandrina perspicillata in northern and Salamandrina terdigitata in southern Italy. Parental lineages diverged at least 10 million years ago during the Lower Pliocene and represent the most ancient split between any congeneric amphibian species endemic to the Italian peninsula. Analysis of the mitochondrial cytochrome b (Cyt b), nuclear-encoded recombination-activating protein (RAG-1) and propriomelanocortin (POMC) genes of more than 250 individuals from populations of both species and from the contact zone, show clear evidence of ongoing hybridization. Whereas 20% of the individuals from the contact population showed no signs of hybridization for the applied markers, the remainder (80%) were identified as first generation hybrids and backcrossed individuals. Our results suggest that hybridization between these two ancient lineages produces viable and fertile offspring, highlighting the need for research on possible mechanisms that prevent the intermixing and hybridization of parental species on a broader geographical scale.