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Fossil remains of extinct mylodontine sloths have been discovered in numerous localities throughout the American supercontinent, but knowledge of them is still mainly centered on the Pleistocene forms rather than their Neogene relatives. In this contribution, previously unpublished cranial and postcranial materials herein ascribed to Glossotherium chapadmalense (Kraglievich), coming from the Pliocene Chapadmalal Formation (Province of Buenos Aires, Argentina), are presented. These remains are described and compared with those of other mylodontine species from South and North America, providing an exhaustive characterization of cranial morphology in G. chapadmalense and some preliminary insights into its postcranial anatomy. The results have revealed a unique combination of differences and similarities of this enigmatic species with the larger-sized representatives of Glossotherium. In addition, these materials allowed shedding light on the long-standing uncertainties regarding its taxonomic status, as this form has been alternately considered to belong to the genera Glossotherium or Glossotheridium over the last century. As a result, the genus Glossotherium was present, at least, at the Early–Late Pliocene transition in the Pampean region of Argentina, and is recorded by the Upper Pleistocene throughout the entire South American continent. The radiation of the Pleistocene species is associated with a consistent body mass increase that was probably a response to the cooling temperatures and higher predation pressure in South America during the Late Pliocene–Early Pleistocene. These phenomena were probably driven by the stable emergence of the Panamanian Isthmus around 3 Ma and the successive faunal turnover pulses of the Great American Biotic Interchange. [ABSTRACT FROM AUTHOR]