Objective This study aims to analyze the codon usage bias and its main influencing factors in the chloroplast genomes of three Prinsepia species, providing a scientific basis for understanding their codon optimization mechanisms, preference patterns, and phylogenetic relationships.

Methods The chloroplast genome sequences of Prinsepia utilis obtained through sampling and sequencing, along with those of Prinsepia sinensis and Prinsepia uniflora downloaded from the NCBI database, were processed in the Geneious bioinformatics software to filter and remove duplicate and CDS sequences shorter than 300 bp. Subsequently, CodonW 1.4.2 was employed to perform codon usage bias analysis on the screened CDS sequences and identify optimal codons.

Results The GCall content of the three species was 38.45%～39.08%, the effective codon number (ENC) of the chloroplast coding sequence of the genome was distributed between 35.60 and 61.00, the ENC value of protein coding genes was greater than 35, and their codon preference was weak. The three optimal codons shared by three species, the third base is mostly A/U. PR2-Plot analysis showed uneven distribution of genes in the four regions, and effective codon number. ENC-Plot analysis showed that most genes were distributed below the standard curve, and all genes were above the diagonal. In summary, natural selection is the primary factor influencing codon bias in the chloroplast genomes of three species of Prinsepia plants.

Conclusion Codon bases of the chloroplast genome from three Prinsepia plants species are mostly ended with A/U. The preference is weak and the main influencing factor is natural selection. Three optimal codons, including AAU, GAU, AGU are identified in this study.