Studying Neotropical wild populations is of particular interest. While this region is facing an escalating habitat degradation, it also has remarkable biodiversity levels, whose origin we are only beginning to understand. A myriad of processes might have had idiosyncratic effects on its numerous species. Within the hottest Neotropical biodiversity hotspot, the Atlantic Forest (AF), species and genetic diversities are organized latitudinally, with decreasing diversity levels southwards. Bradypus variegatus, the brown-throated three-toed sloth, was one of the first species observed to present such pattern. Moreover, within AF, B. variegatus populations seem to be geographically isolated and genetically differentiated. Whether AF B. variegatus isolation, differentiation, and loss of genetic diversity are historical or contemporary (anthropogenic-driven), result from species-specific or general historical events, and if this is of conservation concern remains unclear. Here, we combine micro-evolutionary, multilocus, and high-throughput sequencing approaches to detail the processes responsible for the patterns of genetic diversity on B. variegatus populations in AF, and further understand AF biogeographic history. Few studies made use of similar approaches on Neotropical biodiversity. Our results agree with recent re-interpretations on the AF refugia model and support a species-specific refugium in southern AF, characterized by a metapopulation formation. Finally, we present compelling evidences of the need for conservation actions on AF B. variegatus populations, by comparing genetic diversity levels between populations of different Bradypus species. As far as we know, this is the most comprehensive assessment on Bradypus nuclear DNA diversity.