A GeXP multiplex, RT-PCR assay was developed and optimized that simultaneously measures expression of a suite of immune-relevant genes in rainbow trout (Oncorhynchus mykiss), concentrating on tumor necrosis factor and interleukin-1 ligand/receptor systems and acute phase response genes. The dataset includes expression values for drpt, il11a, il1b1, il1b2, il1b3, il1r-like-1(e3-5), il1r-like-1(e9-11), il1r1-like-a, il1r1-like-b, il1r2, saa, tnfa1, tnfa2, tnfa3, tnfrsf1a, tnfrsf1a-like-a, tnfrsf1a-like-b, tnfrsf5, and tnfrsf9. Gene expression was measured at four time-points post-challenge in both a resistant line (ARS-Fp-R) and a susceptible line (ARS-Fp-S) of rainbow trout. In addition, fish body weight, spleen index and the Flavobacterium psychrophilum load are reported. These data are an extension of information presented and discussed in the article "Proinflammatory cytokine and cytokine receptor gene expression kinetics following challenge with Flavobacterium psychrophilum in resistant and susceptible lines of rainbow trout (Oncorhynchus mykiss)" (Kutyrev et al., 2016). Resources in this dataset:Resource Title: Dataset of proinflammatory cytokine and cytokine receptor gene expression in rainbow trout (Oncorhynchus mykiss) measured using a novel GeXP multiplex, RT-PCR assay. File Name: Web Page, url: https://www.sciencedirect.com/science/article/pii/S2352340917300331 Data in Brief data article providing expression values for a suite of immune-relevant genes in rainbow trout (Oncorhynchus mykiss), concentrating on tumor necrosis factor and interleukin-1 ligand/receptor systems and acute phase response genes. Data are available for download as Excel spreadsheet.Resource Software Recommended: Microsoft Excel,url: https://office.microsoft.com/excel/