The differential exploration of natural resources by Drosophila species has effects on fitness, with changes in life history and metabolic traits. There is a lack of research on the variation in these characters in different environments in Neotropical species of Drosophila. The purpose of this study was to evaluate the profile of life history traits, including viability, development time, and dry weight (as a measure of size), as well as the metabolic pools of triglyceride, glycogen, and protein, in populations from the southern and southeastern regions of Brazil of four Neotropical Drosophila species: D. willistoni, of the Sophophora subgenus, and D. mercatorum, D. maculifrons, and D. ornatifrons, which belong to the Drosophila subgenus. Life history and metabolic traits showed interpopulational variation in at least one species. When significant differences in life history parameters occurred, species of the same subgenus presented similar profiles, i.e., southern populations were larger, less viable, and showed longer development time. This was also observed for triglyceride. However, for the other two metabolic pools (glycogen and total proteins), D. maculifrons and D. ornatifrons presented inverse patterns to the other two species, with the highest values in southeastern populations and the lowest in southern populations. These populational variations indicate plasticity of the examined life history traits, which allows distinctive responses to different environmental conditions shared by speciesof the same subgenus. Nevertheless, interspecific comparisons did not reflect phylogenetic relationships, with the highest viability being found for D. willistoni and D. mercatorum, which is probably correlated to the ability of these species to explore a broader variety of habitats. On the other hand, the storage capability of metabolic pools seems to be species specific, determined by the adaptive history to the quality and availability of resources, with D. mercatorum (low) and D. ornatifrons (high) having opposing capacities to store metabolites from their diets.