Abstract: Exploring the codon usage bias and its main influencing factors in the chloroplast genome of Verbascum plants, providing a theoretical basis for the development and optimization of chloroplast genetic engineering in Verbascum plants. The chloroplast genome protein-coding sequences (CDS) of Verbascum thapsus, Verbascum phoeniceum and Verbascum songaricum were downloaded from the NCBI database, and the complete sequence of CDS, excluding duplicates and that less than 300 bp in length, was selected for further analyses using Geneious v.7.1.3 bioinformatics software. Codon W 1.4.2, CUSP and SPSS 26 were used to analyse the effective codon count (ENC), the relative usage of synonymous codons (RSCU), the GC content of bases 1, 2, and 3 of the gene codon (denoted as GC₁, GC₂, and GC₃, respectively) and the average GC content (GC_all), and were subjected to neutral plot analysis and PR2-plot analysis. Multivariate statistics from neutral plot analysis, PR2-plot analysis, and ENC-plot analysis were employed to predict the codon usage preference patterns in the chloroplast genomes of three species of Verbascum and to screen the optimal codons. The results showed that the complete chloroplast genomes of the three species of Verbascum had sequence lengths of 153338 bp, 153348 bp, and 153291 bp, respectively, and the ENC values of the protein-coding genes of the three species of Verbascum exceeded 35, indicating that the codon preference of the chloroplast genomes of the three species of Verbascum was weak; and the average GC content at each position of the codon (GC_all) was 38.31%, 38.00%, 38.00%, respectively. In addition, GC₁ (46.83%) > GC₂ (39.63%) > GC₃ (28.49%) in Verbascum thapsus; similarly, GC₁ (46.12%) > GC₂ (38.40%) > GC₃ (29.50%) in Verbascum phoeniceum; and GC₁ (46.07%) > GC₂ (38.38%) > GC₃ in Verbascum songaricum, indicating that the distribution of GC content was not uniform in different locations of different species. Neutral mapping analysis, ENC-plot analysis, and PR2-plot analysis indicated that natural selection was the primary factor influencing codon preference in the chloroplast genomes of the three Verbascum species. Furthermore, the RSCU and ENC values were used to identify 11, 15, and 11 optimal codons in Verbascum thapsus, Verbascum phoeniceum, Verbascum songaricum, respectively, among which there were nine common optimal codons, namely AUA, UCC, GCC, AAU, GAU, UGC, UGA, CGU, AGU, and most of codons preference ends with A/U.