Dichlorodiphenyltrichloroethane (DDT) metabolism-based resistance in Drosophila melanogaster is a complex metabolic system associated with the transcription of detoxification related genes, ion transport, lipid and sugar metabolism pathways. However, little is known about the differences regarding the proteome of field- and laboratory-selected resistant Drosophila genotypes. We investigated the impact of DDT resistance in the abundant proteome of field- and laboratory- selected resistant Drosophila using a two-dimensional gel electrophoresis DDT reference map. Proteomic profiling was performed in two DDT susceptible genotypes (Canton-S and 91-C) and three DDT resistant lines (Rst(2)DDT(91-R), Rst(2)DDT(Wisconsin) and Rst(2)DDT(Hikone-R)). Protein spots were stained with Coomassie blue and compared using PDQuest software. Selected protein spots were cut out and analyzed using matrix assisted laser desorption-time of flight mass spectrometry. Querying the NCBInr. 10.21.2003 database with mass spectrometric data yielded the identity of 21 differentially translated proteins in Rst(2)DDT(91-R), Rst(2)DDT(Wisconsin) and Canton-S representing proteins putatively involved in biochemical pathways such as glycolysis and gluconeogenesis, the pentose phosphate pathway, the Krebs cycle and fatty acid oxidation. We hypothesize that both strategies are aimed to use of the pentose phosphate pathway to increase glucose utilization while Rst(2)DDT(91-R) relies primarily on glycolysis to produce reduced NADP and increase DDT detoxification. DDT exposure in Canton-S induced six proteins, while four proteins were repressed in Rst(2)DDT(Hikone-R). Our data suggest that insecticide resistance appears to impact different metabolic pathways in Drosophila genotypes selected with the same pesticide (DDT).