The Pampas biome is a subtropical–temperate grassland region that presents striking climatic characteristics, such as temperatures below zero in the winters and up to 40°C in the summers. Hence, long sampling periods are necessary to better understand the structure of Drosophilidae assemblages through current climatic conditions in the heterogeneous landscape of the Pampas. Samples of drosophilids were taken in natural and urban areas during 12 climatic periods. The environmental influence on the structure of assemblages was temporally and spatially analyzed through nonmetric multidimensional scaling, indicator value, and permutational multivariate analysis of variance. The following hypotheses were tested: 1) the structure of drosophilids assemblages vary through different habitats from the same area, and 2) the presence and abundance of Drosophilidae species in a particular environment is regulated by the interaction between the phytophysionomy and climatic traits of each locality. The Pampas assemblages were dominated by a few species, although it is a highly diverse biome with regards to Drosophilidae. The environmental heterogeneity was confirmed as a determinant factor of the structure of assemblages, and it explained 8.86% of the assemblage composition. Nonetheless, the interaction between temperature and humidity was revealed as the most influential environmental component of the Drosophilidae assemblages, and it explained 37.28% of all the recorded variation. Together, environment type and the climatic elements were able to explain 56.45% of the assemblages' variation. Open and close environments, such as grassland and forest patches, respectively, presented a peculiar composition of Drosophilidae assemblages and should be equally considered in the Pampas preservation strategies.