We investigated the global metabolite response to artificial selection for tolerance to stressful conditions such as cold, heat, starvation, and desiccation, and for longevity in Drosophila melanogaster. Our findings were compared to data from other levels of biological organization, including gene expression, physiological traits, and organismal stress tolerance phenotype. Overall, we found that selection for environmental stress tolerance changes the metabolomic (1)H NMR fingerprint largely in a similar manner independent of the trait selected for, indicating that experimental evolution led to a general stress selection response at the metabolomic level. Integrative analyses across data sets showed little similarity when general correlations between selection effects at the level of the metabolome and gene expression were compared. This is likely due to the fact that the changes caused by these selection regimes were rather mild and/or that the dominating determinants for gene expression and metabolite levels were different. However, expression of a number of genes was correlated with the metabolite data. Many of the identified genes were general stress response genes that are down-regulated in response to selection for some of the stresses in this study. Overall, the results illustrate that selection markedly alters the metabolite profile and that the coupling between different levels of biological organization indeed is present though not very strong for stress selection at this level. The results highlight the extreme complexity of environmental stress adaptation and the difficulty of extrapolating and interpreting responses across levels of biological organization.