Drosoph1la ornatifrons of the guarani group (Diptera: Drosophilidae) is found mainly in humid areas of the Atlantic Forest biome, especially in the southern region of Brazil. Historical and contemporary fragmentation events influenced species diversity and distribution in this biome, although the role of paleoclimatic and paleogeographic events remain to be verified. The objective of the present study was to evaluate the demographic structure of D. ornatifrons from collection sites that are remnants of Atlantic Forest in southern Brazil, in order to contribute to the understanding of the processes that affected the patterns of genetic variability in this species. To achieve this goal, we sequenced 51 individuals from nine localities and 64 individuals from six localities for the mitochondrial genes Cytochrome Oxidase I and II, respectively. Our results indicate that D. ornatifrons may have experienced a demographic expansion event from the southernmost locations of its distribution, most likely from those located next to the coast and in fragments of Atlantic Forest inserted in the Pampa biome (South 2 group), towards the interior (South 1 group). This expansion probably started after the last glacial maximum, between 20,000 and 18,000 years ago, and was intensified near the Pleistocene-Holocene transition, around 12,000 years ago, when temperature started to rise. In this work we discuss how the haplotypes found barriers to gene flow and dispersal, influenced by the biogeographic pattern of Atlantic Forest.