Identifying cryptic species is a challenging but critical task for conservation biology because such identifications improve the resolution of taxonomic inventories and increase our ability to precisely assess the biodiversity of environments as well as the conservation status of rare species. To understand the temporal and spatial distribution of the cryptic species contained in the Drosophila willistoni subgroup, we re-examined all of the individuals of this subgroup identified in a previous work that analyzed drosophilids associated with a forest patch in the Brazilian Cerrado from December 2007 to November 2008. Each collection consisted of banana-baited traps distributed in three vertical strata of the forest (0-, 4-, and 8-m heights) at three horizontal positions (edge, transition between edge and river, and river area). Our results revealed that the specimens originally identified as D. willistoni consisted of three cryptic species: D. willistoni (n = 1,344), D. paulistorum (n = 1,001), and D. tropicalis (one male). Most D. willistoni and D. paulistorum specimens were collected during the rainy season on the ground of the edge forest patch. However, D. paulistorum was more temporally and spatially restricted than D. willistoni. Our findings confirm that these three cryptic species of the subgroup D. willistoni differ not only in abundance but also in ecological tolerance. This study contributes to the improved understanding of the Brazilian savanna communities, whose high levels of species richness and endemism are well documented for plants but poorly known for insects.