[Objective]In order to analyze the codon usage bias of the chloroplast genome of four Zygophyllum species, the study analyzed the codon composition and codon usage bias of 40 selected gene-coding sequences and identified the optimal codons.[Methods] The parity preference (PR2-plot) analysis, effective number of codons (NEC-plot), neutrality plot and RSCU cluster analysis of 40 genes from four Zygophyllum species were carried out using CodonW 1.4.2 and Emboss software in this study. [Results]The codon usage patterns in four Zygophyllum species plastomes were similar, and the GC content of codon was followed by GC1> GC2> GC3, the average ENC values were more than 35, and the CAI was 0.17, indicating the chloroplast genome gene expression level of Zygophyllum species was low and a weak codon preference. The results of neutral mapping analysis, ENC-plot selection and PR2-plot analysis showed that the codon usage pattern was affected by natural selection, which played a dominant role. RSCU cluster analysis showed that Z. fabago, Z. obliquum, Z.xanthoxylum and Z. kaschgaricum had 19, 13,14 and 14 optimal codons. By constructing the high and low gene expression library of the target genes, a total of four optimal genes were finally selected using totally the A\U ending.[Conclusion] The codon bases of chloroplast genome from four Zygophyllum species mostly ended with A\U. The codon usage bias of chloroplast genome have a weak codon preference, and be in?uenced by base mutations . Four optimal codons were identi?ed in this study, and the presence of few optimal codons may better understood the codon optimization, preference mechanism and genetic evolution relationship in Zygophyllum.