To study the homology and evolutionary relationship among eIF5A gene of Populus trichocarpa, bioinformatics softwares and websites were used to predict and analyze its base distribution, amino acid composition, hydrophilcity or hydrophobicity, secondary structure and tertiary structure. A polygenetic tree was constructed based on the corresponding eIF5A proteins from other species, and the conserved sequences in these proteins were analyzed. The results showed that four eIF5A genes were located on different chromosomes and all had five exons. The research still found that the amino acid number and sequence hydrophobic in eIF5A gene family presented some differences. Protein subcellular localization analysis showed that all members of eIF5A were located in the cytoplasm. Furthermore, the main composition of 4 amino acid sequences were random coil, extended strand and alpha helix via secondary structure prediction, and they were similar to each other in three-level structure. The results of this investigation could definitely provide some foundation for further function analysis of eIF5A gene family.