The fruit fly Drosophila melanogaster is a model organism to study several aspects of metazoan biology. Most of the work has been conducted in adult fruit flies, including laboratory and field-derived specimens, but Drosophila melanogaster larvae recently became a valuable model to better understand animal physiology, development or host-microbe interactions. While adult flies can be easily assigned to a given Drosophila species based on morphological characteristics, such visual identification is more intricate at the larval stage. This could explain the limited number of studies focusing on larvae, especially field-derived samples. Here, we developed a Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) assay that discriminates D. melanogaster from other ecologically relevant Drosophila species at the larval stage. The method, which targets the cytochrome oxidase I (COI) gene, was validated using laboratory-derived larvae from seven D. melanogaster populations originating from different geographic areas as well as six Drosophila species. We further validated this PCR-RFLP assay in a natural context, by identifying wild larvae collected in two locations in France. Notably, among all PCR-RFLP profiles that matched the D. melanogaster species, 100% were correctly identified, as confirmed by COI sequencing. In summary, our work provides a rapid, simple and accurate molecular tool to identify D. melanogaster from field-collected larvae.