Saber-toothed cats (Machairodontinae) are among the most widely recognized representatives of the now largely extinct Pleistocene megafauna. However, many aspects of their ecology, evolution, and extinction remain uncertain. Although ancient-DNA studies have led to huge advances in our knowledge of these aspects of many other megafauna species (e.g., mammoths and cave bears), relatively few ancient-DNA studies have focused on saber-toothed cats [ 1–3 ], and they have been restricted to short fragments of mitochondrial DNA. Here we investigate the evolutionary history of two lineages of saber-toothed cats (Smilodon and Homotherium) in relation to living carnivores and find that the Machairodontinae form a well-supported clade that is distinct from all living felids. We present partial mitochondrial genomes from one S. populator sample and three Homotherium sp. samples, including the only Late Pleistocene Homotherium sample from Eurasia [ 4 ]. We confirm the identification of the unique Late Pleistocene European fossil through ancient-DNA analyses, thus strengthening the evidence that Homotherium occurred in Europe over 200,000 years later than previously believed. This in turn forces a re-evaluation of its demography and extinction dynamics. Within the Machairodontinae, we find a deep divergence between Smilodon and Homotherium (∼18 million years) but limited diversity between the American and European Homotherium specimens. The genetic data support the hypothesis that all Late Pleistocene (or post-Villafrancian) Homotherium should be considered a single species, H. latidens, which was previously proposed based on morphological data [ 5, 6 ].