Abstract Background Cold acclimation is the process by which plants adapt to the low, non freezing temperatures that naturally occur during late autumn or early winter. This process enables the plants to resist the freezing temperatures of winter. Temperatures similar to those associated with cold acclimation are also used by the fruit industry to delay fruit ripening in peaches. However, peaches that are subjected to long periods of cold storage may develop chilling injury symptoms (woolliness and internal breakdown). In order to better understand the relationship between cold acclimation and chilling injury in peaches, we isolated and functionally characterized cold-regulated promoters from cold-inducible genes identified by digitally analyzing a large EST dataset. Results Digital expression analyses of EST datasets, revealed 164 cold-induced peach genes, several of which show similarities to genes associated with cold acclimation and cold stress responses. The promoters of three of these cold-inducible genes (Ppbec1, Ppxero2 and Pptha1) were fused to the GUS reporter gene and characterized for cold-inducibility using both transient transformation assays in peach fruits (in fruta) and stable transformation in Arabidopsis thaliana. These assays demonstrate that the promoter Pptha1 is not cold-inducible, whereas the Ppbec1 and Ppxero2 promoter constructs are cold-inducible. Conclusion This work demonstrates that during cold storage, peach fruits differentially express genes that are associated with cold acclimation. Functional characterization of these promoters in transient transformation assays in fruta as well as stable transformation in Arabidopsis, demonstrate that the isolated Ppbec1 and Ppxero2 promoters are cold-inducible promoters, whereas the isolated Pptha1 promoter is not cold-inducible. Additionally, the cold-inducible activity of the Ppbec1 and Ppxero2 promoters suggest that there is a conserved heterologous cold-inducible regulation of these promoters in peach and Arabidopsis. These results reveal that digital expression analyses may be used in non-model species to identify candidate genes whose promoters are differentially expressed in response to exogenous stimuli.