Abstract: To investigate the codon usage pattern and factors influencing the chloroplast codons of Polyspora chrysandra, 42 CDSs were screened and analyzed using CodonW and CUSP online programs. The results showed that the GC content of chloroplast codons in the chloroplast genome of P. chrysandra varied among positions, with preference ending in A/U; the ENC values ranged from 35.64 to 53.19, with weak codon preference; there was a significant positive correlation between codon number and GC₃ and ENC, and codon preference was related to gene length. The results of the neutral mapping analysis, parity preference analysis and effective codon mapping analysis show that natural selection has an important influence on codon usage patterns; 19 optimal codons further demonstrate that codon preference ends with A/U; the CDS phylogenetic tree is biased towards the construction of species evolutionary relationships, and RSCU clustering focuses on the mining of specific evolutionary patterns of codons, which can provide important data for inferring the similarity and evolutionary relationships among species and genes. The study provides useful information for the evolutionary characterization of codons and chloroplast genetic engineering research of P. chrysandra.