Sloths (Xenarthra, Folivora) were one of the dominant mammalian groups in Southern and Central America until the early Holocene. Research into the molecular evolution and biology of sloths, previously limited to extant species, has started to leverage information contained in the large recent fossil record of this group through ancient DNA methods. Cueva del Milodón (Ultima Esperanza, Chile) is renowned for its exceptionally preserved faunal record. Remains of Darwin’s ground sloth (Mylodon darwinii) are found there from the end of the last Ice Age to their extinction in the early Holocene. Here I present novel findings from genomic data from multiple M. darwinii individuals, exploring genetic diversity in this site. Using ancient DNA methods, nine new mitochondrial genomes and two nuclear genomes were recovered from bone, skin and coprolites from Cueva del Milodón in British and Swiss museum collections.