Abstract <p>Codon usage bias (CUB) is a unique characteristic of genomes which refers to non-random usage of selected synonymous codons in the genes. Lamiaceae, the largest family of order lamiales, is significant to humans as its herbaceous plants are useful for flavor, fragrance and, most importantly, medicinal properties. In the present study, the codon usage patterns and variation sources of the chloroplast genomes of <i>Plectranthus barbatus</i> and allied species were systematically analyzed. Our results showed that the chloroplast genomes of five <i>Plectranthus</i> plants end with A/T(U) bases, and a total of 24 optimal codons were detected among them. Effective Number of codons vs. GC content of third codon positions (ENC-GC3s analysis), Parity Rule 2 (PR2) analysis, and neutrality plot analysis showed that the codon bias of the <i>Plectranthus</i> plants was influenced by many factors, natural selection being the key influencing factor. Collectively, this study aims to highlight the shortage of chloroplast sequencing data of genus <i>Plectranthus</i>; and a consistent pattern of CUB in the plastomes of five <i>Plectranthus</i> plants, influenced by a combination of natural selection, mutation pressure and possibly other unidentified factors. Significant correlation was found between nucleotide composition parameters, gene expression as well as protein properties with synonymous codon usage order (SCUO) of cp genes which supported the finding that expression of cp genes was associated with the degree of CUB. The results may also shed light on the way to improve the expression efficiency of transgenes in <i>Plectranthus</i> plants based on codon optimization.</p>