The mountains in the Atlantic Forest domain are environments that harbor a high biodiversity, including species adapted to colder climates that were probably influenced by the climatic variations of the Pleistocene. To understand the phylogeographic pattern and assess the taxonomic boundaries between two sister montane species, a genomic study of the butterflies Actinote mantiqueira and A. alalia (Nymphalidae: Acraeini) was conducted. Analyses based on partial sequences of the mitochondrial gene COI (barcode region) failed to recover any phylogenetic or genetic structure discriminating the two species or sampling localities. However, single nucleotide polymorphisms gathered using Genotyping-by-Sequencing provided a strong isolation pattern in all analyses (genetic distance, phylogenetic hypothesis, clustering analyses, and F ST statistics) which is consistent with morphology, separating all individuals of A. alalia from all populations of A. mantiqueira. The three sampled mountain ranges where A. mantiqueira populations occur-Serra do Mar, Serra da Mantiqueira, and Poços de Caldas Plateau-were identified as three isolated clusters. Paleoclimate simulations indicate that both species' distributions changed according to climatic oscillations in the Pleistocene period, with the two species potentially occurring in areas of lower altitude during glacial periods when compared to the interglacial periods (as the present). Besides, a potential path between their distribution through the Serra do Mar Mountain range was inferred. Therefore, the Pleistocene climatic fluctuation had a significant impact on the speciation process between A. alalia and A. mantiqueira, which was brought on by isolation at different mountain summits during interglacial periods, as shown by the modeled historical distribution and the observed genetic structure.