The ability of an organism to respond to nutritional stress can be a plastic character under the action of natural selection, affecting several characteristics, including life history and energy storage. The Drosophila genus (Diptera; Drosophilidae) presents high variability regarding natural resource exploration. However, most works on this theme have studied the model species D. melanogaster (J.W.Meigen) and little is known about Neotropical species. Thus, the current work evaluated the effects of three diets, with different carbohydrate:protein ratios, on life history (viability and development time) and metabolic pools (triglycerides, glycogen, and total soluble protein contents) of three Neotropical species. Our results showed that Drosophila willistoni (A.H.Sturtevant) (Sophophora subgenus) was viable on all diets, while D. maculifrons (O.Duda) (Drosophila subgenus) was not, and D. ornatifrons (O.Duda) (Drosophila subgenus) was barely viable on the sugary diet. This diet increased the development time of D. willistoni and D. ornatifrons, and D. willistoni glycogen content. Thus, the viability of D. maculifrons and D. ornatifrons seems to depend on a certain amount of protein and/or a low concentration of carbohydrate in the diet. A more evident effect of the diets on triglyceride and protein pools was detected in D. ornatifrons, which could be related to its capacity to explore dung and carrion as food resource in nature. The results found demonstrated that the evolutionary history and differential adaptations to natural macronutrient resources are important to define the amplitude of response that a species can present when faced with dietary variation.