In the wild, larvae of several species of Drosophila develop in heterogeneous and rapidly changing environments sharing resources as food and space. In this scenario, sensory systems contribute to detect, localize and recognize congeners and heterospecifics, and provide information about the availability of food and chemical features of environments where animals live. We investigated the behavior of D. simulans and D. buzzatii larvae to chemicals emitted by conspecific and heterospecific larvae. Our goal was to understand the role of these substances in the selection of pupation sites in the two species that cohabit within decaying prickly pear fruits (Opuntia ficus-indica). In these breeding sites, larvae of D. simulans and D. buzzatii detect larvae of the other species changing their pupation site preferences. Larvae of the two species pupated in the part of the fruit containing no or few heterospecifics, and spent a longer time in/on spots marked by conspecifics rather than heterospecifics. In contrast, larvae of the two species reared in isolation from conspecifics pupated randomly over the substrate and spent a similar amount of time on spots marked by conspecifics and by heterospecifics. Our results indicate that early chemically-based experience with conspecific larvae is critical for the selection of the pupation sites in D. simulans and D. buzzatii, and that pupation site preferences of Drosophila larvae depend on species-specific chemical cues. These preferences can be modulate by the presence of larvae of the same or another species.