Abstract: The analysis of codon usage patterns and their main influencing factors in four species of Bletilla Rchb. lays a solid theoretical foundation for a deeper understanding of the genetic characteristics, environmental adaptability, genetic improvement, and variety cultivation strategies of Bletilla Rchb. plants. The neutrality plot, ENC-plot, and PR2-plot analysis of chloroplast genome codons in 4 Bletilla Rchb. plants of Bletilla striata, Bletilla formosana, Bletilla sinensis, Bletilla ochracea were conducted based on CUSP, Codon W 1.4.2 and EMBOSS software. The study revealed that the four optimal codons shared by these four species all exhibit the characteristic that the third base is A/U, and the effective number of codons (ENC) of all protein-coding genes exceeds the threshold of 35. This phenomenon reflects a relatively weak trend in codon preference in Lepidoptera species. Furthermore, through parity preference (PR2-Plot) analysis, we observed that the distribution of genes in the four regions is not balanced; and effective codon number analysis (ENC-Plot) showed that most genes are located below the standard curve. Furthermore, in the neutral plot analysis, all genes were significantly shifted upward from the diagonal. This series of analyzes collectively shows that codon usage bias is affected by both mutation and natural selection, but natural selection plays a more critical role in this process and has a more significant impact on codon preference. In summary, this study provides a comprehensive and in-depth analysis of the codon usage bias in the chloroplast genomes of four species of the genus Bryoides, and clearly points out that natural selection is the dominant factor in shaping their codon preferences. This discovery not only enriches our understanding of the genetic characteristics of Albus genus plants, but also lays a solid theoretical foundation for subsequent exploration of their environmental adaptability, genetic improvement and variety breeding strategies.