Atropa belladonna is a valuable medicinal plant and a commercial source of tropane alkaloids, which are frequently utilized in therapeutic practice. In this study, bioinformaticmethodologies were used to examine the pattern of coding sequences and the factors thatmight influence codon usage bias in the chloroplast genome of Atropa belladonna andother nightshade genomes. The chloroplast engineering being a promising field in modernbiotechnology, the characterization of chloroplast genome is very important. The resultsrevealed that the chloroplast genomes of Nicotiana tabacum, Solanum lycopersicum, Capsicum frutescens, Datura stramonium, Lyciumbarbarum, Solanum melongena, and Solanumtuberosum exhibited comparable codon usage patterns. In these chloroplast genomes, weobserved a weak codon usage bias. According to the correspondence analysis, the genesisof the codon use bias in these chloroplast genes might be explained by natural selection,directed mutational pressure, and other factors. GC12 and GC3S were shown to have nomeaningful relationship. Further research revealed that natural selection primarily shapedthe codon usage in A. belladonna and other nightshade genomes for translational efficiency. The sequencing properties of these chloroplast genomes were also investigated by investing the occurrences of palindromes and inverted repeats, which would be useful forfuture research on medicinal plants.