Stressors inherent in aquaculture production can induce physiological responses which can have adverse effects on growth, reproduction and immunocompetence. We developed a reference transcriptome to evaluate responses to heat shock, low temperature, high salinity, crowding, and low dissolved oxygen/high carbon dioxide. The reference sequence was obtained by Roche 454 pyrosequencing of a normalized cDNA library and is therefore solely qualitative, containing representative samples from all the different stressors (including controls) and tissues (gill, brain, liver, spleen, kidney and muscle). A total of 3,160,306 high quality reads were generated and 2,708,437 reads were assembled into 110,026 contigs. Comparisons with existing transcriptome data reveal a significant contribution to publicly available transcriptome data for rainbow trout. This resource will be used in future studies for global characterization stress responses at the level of the transcriptome.