Codon usage is the selective and nonrandom use of synonymous codons to encode amino acids in genes for proteins. The analysis of codon usage may improve the understanding of cocion preferences between different species and allow to rebuild the codons of exogenous genes to increase the expression efficiency of exogenous genes, Here, codon DNA sequence (CDS) of four poplar species, including Populus tremuloides Michx., P. tomentosa Carr., P. deltoides Marsh., and P. trichocarpa Torr. & Gray., is used to analyze the relative frequency of synonymous codon (RFSC). High-frequency codons are selected by high-frequency (HF) codon analysis. The results indicate that the codon usage is common for all four poplar species and the codon preference is quite similar among the four poplar species. However, CCT encoding for Pro, and ACT coding for Thr are the preferred codons in P. tremuloides and P. tomentosa, whereas CCA coding for Pro, and ACA coding for Thr are preferred in P. deltoides and P. trichocarpa The codons such as TGC coding for Cys, TTC coding for Phe, and AAG coding for Lys, are preferred in the poplar species except P trichocarpa. GAG coding for Glu is preferred only in P deltoides, while the other three poplar species prefer to use GAA. The commonness of preferred codon allows exogenous gene designed by the preferred cocion of one of the different poplar species to be used in other poplar species.