Abstract: To clarify the usage pattern of codons in the chloroplast genome of Pleione D. Don, 16 species of Pleione D. Don were used as study materials, and Codon W 1.4.2 and online software CUSP were used to obtain the parameters of GC, RSCU, ENC of codons in Pleione D. Don, etc., and neutral mapping, PR2-plot and optimal codon analysis were also carried out. The results showed that the contents of codons GC₁, GC₂, GC₃ and GC_all were similar in 16 species, and each species showed the following pattern: GC₁>GC₂>GC₃, which indicated that the GC contents in the same species of Pleione D. Don were not uniformly distributed in different positions of the codons, and the codons preferred to end in A/U; the ENC values were all more than 35, which indicated that the codon preferences of Pleione D. Don were weak. The r QW69esults of neutral mapping, parity preference analysis and effective codon mapping showed that the formation of codon preferences in the chloroplast genomes of Pleione D. Don was mainly influenced by natural selection. The relative sequence usage(RSCU) and ENC values were used to identify the 10～22 optimal codons, most of which ended in A/U. The present study provides useful information on the evolutionary characteristics of codons in Pleione D. Don and chloroplast genetic engineering.