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Rainbow trout (Oncorhynchus mykiss) are widely cultured throughout the word for commercial aquaculture. However, as a cold-water species, rainbow trout are highly susceptible to heat stress, which may cause pathological signs or diseases by alleviating the immune roles and then lead to mass mortality. Understanding the molecular mechanisms that occur in the rainbow trout in response to heat stress will be useful to decrease heat stress-related morbidity and mortality in trout aquaculture. In the present study, we conducted transcriptome analysis of head kidney tissue in rainbow trout under heat-stress (24 °C) and control (18 °C) conditions, to identify heat stress-induced genes and pathways. More than 281 million clean reads were generated from six head kidney libraries. Using an adjusted P-value of P < 0.05 as the threshold, a total of 443 differentially expressed genes (DEGs) were identified, including members of the HSP90, HSP70, HSP60, and HSP40 family and several cofactors or cochaperones. The RNA-seq results were confirmed by RT-qPCR. Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis of DEGs were performed. Many genes involved in maintaining homeostasis or adapting to stress and stimuli were highly induced in response to high temperature. The most significantly enriched pathway was “Protein processing in endoplasmic reticulum (ER)”, a quality control system that ensures correct protein folding or degradation of misfolded polypeptides by ER-associated degradation. Other signaling pathways involved in regulation of immune system and post-transcriptional regulation of spliceosome were also critical for thermal adaptation. These findings improve our understanding of the molecular mechanisms of heat stress responses and are useful to develop strategies for the improvement of rainbow trout survival rate during summer high-temperature period.Evaluation of rainbow trout head kidney transcriptome under heat-stress.Heat stress induces heat shock proteins, cofactors, and cochaperones in rainbow trout head kidney.Heat stress modulates pathways including immune system, protein metabolism, and spliceosome in rainbow trout head kidney.