In 1937 a right hominin femur shaft with archaic morphology was excavated from the cave of Hohlenstein-Stadel in the Swabian Jura of southwestern Germany. The specimen was discovered in a layer corresponding to the Middle Paleolithic. Attempts to directly date the femur were inconsistent and indicated that the bone may be out of range for radiocarbon dating. Here we present genetic analyses of the femur shaft in order to assess the age and phylogenetic position of this ancient hominin bone. Hybridization capture in combination with next generation sequencing were used to reconstruct the complete mitochondrial genome (mtDNA). A phylogenetic comparison with modern human, Denisovan and an extended dataset of Neandertal mtDNA sequences revealed a closer relationship of the femur’s mtDNA to Neandertals. The Hohlenstein-Stadel mtDNA falls, however, basal to all other Neandertal individuals displaying the deepest divergence and a short phylogenetic branch length. Those results indicate an age for the hominin femur notably older than previously suggested. Using a Bayesian statistic framework we performed a molecular dating to identify the temporal range of the Hohlenstein-Stadel femur and provide important insights into the mitochondrial diversity of the Neandertal populations through the Late Pleistocene.