Thermal stress can pose a major challenge to salmonid fish. A 4x44K oligonucleotide microarray approach was used to screen for genetically determined variations of a temperature stress response during acclimation in fish gills, a highly specialized and complex organ responsible for gas and electrolyte exchange as well as excretion. The comparison addressed transcriptional changes in the local breeding strain BORN and imported (TCO) rainbow trout after graded 2-week acclimation to 8 and 23 °C. Besides well-characterized mediators of thermoregulation such as genes encoding cold-inducible RNA-binding protein and heat shock proteins, the present microarray study suggests several new candidate genes commonly regulated in gills of the two trout lines. Having identified the differential expression of thermoregulated genes as duplicated paralogues, they were subsequently validated in a gill cell model. Moreover, the comparison of transcriptome profiles provides evidence for distinctively employed expression patterns. The induction of genes encoding factors of the early innate immunity in BORN trout upon warming contrasts with the increased expression of adaptive immune genes in import trout. Cold acclimation induced genes assigned to the functional categories "cell death" and "ion channel activity" in import trout, but repressed "lipid metabolism." This manuscript provides an overview of the genes of the multifunctional gills in rainbow trout that are mandated after temperature change, suggesting links between the different temperature-dependent pathways and gene networks.