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Reference genes are critical to obtain reliable results of reverse transcription real-time quantitative PCR (RT-qPCR), which is widely used for relative quantification of gene expression. In this study, we evaluated the validity of seven candidate reference genes for normalization in RT-qPCR analysis in the deep-sea bacterium Shewanella psychrophila WP2 under different environmental conditions. Among the set of genes investigated, gyrA, 16S rRNA and rho were identified as the most suitable reference genes for WP2 at different temperatures, hydrostatic pressures and salinities, respectively. Notably, the rho gene is conserved in Shewanella genus and other deep-sea bacteria, thus, could be used as a versatile reference gene for RT-qPCR analysis of these microorganisms under extreme environmental conditions.