Abstract: Metal chaperones play key roles in plant physiological activities, and could bind metal ions and transport them to target proteins in order to maintain the metal ions homeostasis of cells. Currently, the properties and function analysis of the metal chaperones HIPP (heavy metal-associated isoprenylated plant protein) is mostly limited to the model plant Arabidopsis thaliana. In this study, 14 PtHIPPs were identified in Populus trichocarpa, all of which had two conserved metal ion binding motif MXCXXC, as well as the conserved isoprenylation motif in the carboxyl terminal. The amino terminal of PtHIPPs was form the βαββαβ secondary structure. Some PtHIPPs had lysine-rich and/or glycine-rich regions. PtHIPPs proteins could be divided into three subgroups according to gene structure, protein conservation motifs and phylogenetic analysis. The tissue-specific expression data of PlantGenIE and the results of fluorescence quantitative PCR identification showed that PtHIPPs genes of P.trichocarpa and PnHIPPs genes of Populus simonii×Populus nigra had tissue-specific expression characterizations and differences. In addition, the expression patterns of PnHIPPs in different tissues of P.simonii×P.nigra seedlings were changed after treated with copper deficiency or copper excess conditions for different times. This study provided theoretical groundwork for identifying the roles of HIPPs proteins in maintaining copper homeostasis in woody model plant Populus.