Insects in the field are exposed to multiple bouts of cold, and there is increasing evidence that the fitness consequences of repeated cold exposure differ from the impacts of a single cold exposure. We tested the hypothesis that different kinds of cold exposure (in this case, single short, prolonged and repeated cold exposure) would result in differential gene expression. We exposed 3 day old adult female wild-type Drosophila melanogaster (Diptera: Drosophilidae) to –0.5°C for a single 2 h exposure, a single 10 h exposure, or five 2 h exposures on consecutive days, and extracted RNA after 6 h of recovery. Global gene expression was quantified using an oligonucleotide microarray and validated with real-time PCR using different biological replicates. We identified 76 genes upregulated in response to multiple cold exposure, 69 in response to prolonged cold exposure and 20 genes upregulated in response to a single short cold exposure, with a small amount of overlap between treatments. Three genes – Turandot A, Hephaestus and CG11374 – were upregulated in response to all three cold exposure treatments. Key functional groups upregulated include genes associated with muscle structure and function, the immune response, stress response, carbohydrate metabolism and egg production. We conclude that cold exposure has wide-ranging effects on gene expression in D. melanogaster and that increased duration or frequency of cold exposure has impacts different to those of a single short cold exposure. This has important implications for extrapolating laboratory studies of insect overwintering that are based on only a single cold exposure.