• Codons are essential for transmitting genetic information, and the phenomenon of codon usage bias (CUB) arises when species or genes show preferences for specific synonymous codons. Cymbidium plants, play a significant role in biodiversity conservation and evolutionary research. China, particularly Guizhou Province, is rich in Cymbidium populations. However, studies on codon characteristics in Cymbidium species are limited. • This study comprehensively analyzes of CUB in the chloroplast genomes of 12 Cymbidium species. The findings highlight the importance of natural selection in shaping CUB preferences and emphasize the significance of codon optimization for efficient gene expression. The results contribute to our understanding of Cymbidium genome evolution and have implications for genetic engineering research, resource identification, and exogenous gene expression in Cymbidium plants. Cymbidium plants play a vital role in biodiversity conservation, evolutionary research, and biological protection. Understanding codon usage bias (CUB) in Cymbidium plants is crucial for unraveling the mechanism of species evolution and gene expression. In this study, we systematically analyzed the CUB and identified sources of variance in chloroplast genomes of 12 Cymbidium species to explore the main factors influencing CUB. Using CodonW 1.4.2 software and CUSP program, we screened 52 coding DNA sequences (CDS) within the complete chloroplast genome sequences of the twelve Cymbidium species. Subsequently, neutral, ENC-plot, and PR2-plot mapping analyses were performed to explore the relationships between code usage and genetic elements. The results revealed a consistent bias for guanine-cytosine (GC) bases at the first and second positions (GC1 > GC2 > GC3) of chloroplast genome codons in all 12 Cymbidium species. Moreover, the third base of codons exhibited a predilection for adenine/thymine (A/T) or uracil (U). The effective number of codons (ENC) ranged from 48.6 % to 49.49 %, with an average of 49.07, indicating a weak CUB. Additionally, codon adaptation index (CAI) ranged from 0.167 to 0.171, further supporting the weak CUB. The mapping analyses consistently highlighted natural selection as the primary factor influencing CUB in the 12 orchid chloroplast genomes while acknowledging the impact of other factors, such as mutations. Through optical codon analysis, we identified the top ten preferred codons (UUA, AUU, UCU, CCU, ACU, GCU, AAA, CGA, AGU, and GGU), all of which ended with A or U. These findings provide valuable insights for future studies involving codon modification of foreign genes and the development of high-expression transgenic techniques in Cymbidium plants. [ABSTRACT FROM AUTHOR]