In this study, we quantified the three pivotal genetic processes (i.e., genetic diversity, spatial genetic structuring and migration) necessary for a better biological understanding and management of the singular “living-fossil” and near-threatened mouse opossum marsupial Dromiciops gliroides, the “Monito del Monte”, in south-central Chile. We used 11 microsatellite loci to genotype 47 individuals distributed on the mainland and northern Chiloé Island. Allelic richness, observed and expected heterozygosity, inbreeding coefficient and levels of genetic differentiation were estimated. The genetic structure was assessed based on Bayesian clustering methods. In addition, potential migration scenarios were evaluated based on a coalescent theory framework and Bayesian approach to parameter estimations. Microsatellites revealed moderate to high levels of genetic diversity across sampled localities. Moreover, such molecular markers suggested that at least two consistent genetic clusters could be identified along the D. gliroides distribution (“Northern” and “Southern” cluster). However, general levels of genetic differentiation observed among localities and between the two genetic clusters were relatively low. Migration analyses showed that the most likely routes of migration of D. gliroides occurred a) from the Southern cluster to the Northern cluster and b) from the Mainland to Chiloé Island. Our results could represent critical information for future conservation programs and for a recent proposal about the taxonomic status of this unique mouse opossum marsupial.