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Glutathione peroxidases (GPXs) are important enzymes in the glutathione-ascorbate cycle for catalyzing the reduction of H₂O₂ or organic hydroperoxides to water. GPXs play an essential role in plant growth and development by participating in photosynthesis, respiration, and stress tolerance. <i>Rhodiola crenulata</i> is a popular traditional Chinese medicinal plant which displays an extreme energy of tolerance to harsh alpine climate. The GPXs gene family might provide <i>R. crenulata</i> for extensively tolerance to environment stimulus. In this study, five GPX genes were isolated from <i>R. crenulata</i>. The protein amino acid sequences were analyzed by bioinformation softwares with the results that <i>RcGPXs</i> gene sequences contained three conserve cysteine residues, and the subcellular location predication were in the chloroplast, endoplasmic reticulum, or cytoplasm. Five <i>RcGPXs</i> members presented spatial and temporal specific expression with higher levels in young and green organs. And the expression patterns of <i>RcGPXs</i> in response to stresses or plant hormones were investigated by quantitative real-time PCR. In addition, the putative interaction proteins of <i>RcGPXs</i> were obtained by yeast two-hybrid with the results that <i>RcGPXs</i> could physically interact with specific proteins of multiple pathways like transcription factor, calmodulin, thioredoxin, and abscisic acid signal pathway. These results showed the regulation mechanism of <i>RcGPXs</i> were complicated and they were necessary for <i>R. crenulata</i> to adapt to the treacherous weather in highland.