Drosophila antoneitae is a cactophilic species, ovipositing primarily in the host cacti Cereus hildmaniannus. Both species are found in drained soils or rocky outcrops in the Missiones region-one of the Seasonally Dry Tropical Forest (SDTF) nucleis, and in south Brazilian coast. The Missiones nuclei comprises the basin of the Paraná-Paraguai Rivers and it is a possible area of climatic stability during the Quaternary oscillations. To evaluate the current distribution of the genetic diversity of D. antoneitae and its association with landscape modifications, the following analyses were performed: populational structure, establishment of phylogeographic hypotheses and demographic events, neutrality tests and paleoecological niche modeling. The mitochondrial gene COI and the nuclear gene period were analyzed. The gene period had low genetic diversity and an homogeneity on the distribution of genetic variability. For gene COI analysis we found a high polymorphism and genetic structure among populations, forming three groups: one in Santa Catarina and Rio Grande do Sul, another in Paraná and São Paulo and the third in the midwestern Brazil. The midwestern population is probably the oldest one, coinciding with the most climatically stable area of this study. This group forms a network of separate haplotypes, due to a high structuring and isolation, probably with Paraná River acting as major barrier for gene flow. We suggest that the possible center of dispersion of the remaining populations of D. antonietae is located in southeastern Brazil, and there were several migration events to other regions of its distribution. There is evidence of shared polymorphism due to recent diversification of populations. Both gene clusters exhibit signs of population expansion, especially in border areas at the Missiones nuclei, where the climate seems less stable. The demographic expansion period is recent and coincides with the major expansion of dry vegetation, which can also play a critical role in structuring populations.