The temperate rainforests of southern Chile, a recognized biodiversity hotspot, were significantly affected by Pleistocene glacial cycles in their southern portion and have been severely disrupted mainly due to recent human activities. Additionally, the landscape is characterized by a series of potential barriers to gene flow, such as the Chacao Channel, Cordillera de Piuche in Chiloé and both the Ancud and the Corcovado gulfs. We used mitochondrial DNA sequences and microsatellite data across several populations to evaluate the genetic variability and structure of the sigmodontine rodent Abrothrix olivacea brachiotis, one of the most common species of small mammals and an inhabitant of these biodiverse forests. Sequencing data showed that along with the recovery of high haplotype variation for this species, there was a low nucleotide diversity between haplotypes, showing no genetic differences between the Chiloé Island and continental populations in southern Chile or through any other geographic barrier in the study area. However, microsatellite data exhibited some level of population structuring. The most evident clusterings were those of the Chiloé Island and that of North Patagonia. These findings are corroborated by a barrier analysis that showed a genetic barrier in the latter areas, whereas the Chacao Channel was not a significant barrier for this rodent. Overall, the genetic variability and structure of A. o. brachiotis was concordant with historical factors, such as the Last Glacial Maximum and the presence of geographic elements that isolate populations.