The Apennine chamois (Rupicapra cf pyrenaica) is a very endangered mountain mammal. At the beginning of the 20th century, only a small population survived in the Abruzzo, Lazio and Molise National Park (Central Italy) and, despite its reintroduction in different Apennine massifs and an increased census size, its genetic variability is the lowest among bottlenecked mammals. The ancient DNA analysis of a skull dated back to ~ 3000 cal yr BP allowed us to describe a new haplotype belonging to the mitochondrial Central Clade (including Chartreuse and Apennine populations) but never found in extant chamois. This result underlines that the demographic collapse of Apennine populations, which probably started in the Pleistocene, was combined with an ever-increasing genetic erosion in gradually smaller and isolated populations.