AbstractThe insect cuticle is the interface between internal homeostasis and the often harsh external environment. Cuticular hydrocarbons (CHCs) are key constituents of this hard cuticle and are associated with a variety of functions including stress response and communication. CHC production and deposition on the insect cuticle vary among natural populations and are affected by developmental temperature; however, little is known about CHC plasticity in response to the environment experienced following eclosion, during which time the insect cuticle undergoes several crucial changes. We targeted this crucial to important phase and studied post‐eclosion temperature effects on CHC profiles in two natural populations of Drosophila melanogaster. A forty‐eight hour post‐eclosion exposure to three different temperatures (18, 25, and 30°C) significantly affected CHCs in both ancestral African and more recently derived North American populations of D. melanogaster. A clear shift from shorter to longer CHCs chain length was observed with increasing temperature, and the effects of post‐eclosion temperature varied across populations and between sexes. The quantitative differences in CHCs were associated with variation in desiccation tolerance among populations. Surprisingly, we did not detect any significant differences in water loss rate between African and North American populations. Overall, our results demonstrate strong genetic and plasticity effects in CHC profiles in response to environmental temperatures experienced at the adult stage as well as associations with desiccation tolerance, which is crucial in understanding holometabolan responses to stress.