<p>Plant HKTs (High-affinity K⁺ transporters) are essential transporters for ion transport and homeostasis and play crucial roles in plant growth and stress responses. However, the evolution of HKTs in <i>Populus</i> species and their functions require further investigation. In this study, we identified 16 HKTs from six <i>Populus</i> species. All poplar HKTs were classified as Class I HKTs because of their physiological relationships and the conservation of amino acids in key structures, which aligns with their conserved evolutionary coding sequences. The analysis of the protein domains, motifs and gene structures of 16 poplar HKTs revealed consistent conservation, with the exception of two members. The number of homologs and their chromosome locations indicated the differentiation of HKTs during poplar evolution and adaptation. Poplar HKTs can be classified into two subgroups on the basis of their physiological relationships and distinct protein structures. Gene expression pattern analysis revealed that poplar HKTs presented relatively high expression levels in roots and stems under salt stress. Furthermore, cis-element analysis and protein interaction predictions provide insights into the functions of HKTs under salt stress through the activation of ion transporters, proline content, and ATPases regulated by hormonal signals and MYB transcription factors. In conclusion, our research established a theoretical framework for investigating the evolutionary relationships and functional roles of HKTs in <i>Populus</i> species and offered valuable insights into the functions and underlying mechanisms of poplar HKTs in specific tissues under various stress conditions.</p>