Abstract: Codon usage bias defines the phenomenon of different utilization frequency of the synonymous codon,and this phenomenon widely exists in various plants. In the present study, 45 coding DNA sequences（CDS） in the chloroplast genome of Pinus kesiya var. langbianensis were analyzed using Codon W 1.4.2 and online software CUSP. The results revealed that the GC content of the third base in the codons of chloroplast genome of P. kesiya var. langbianensis was 29.63%, the third base of codons preferred to AT; ENC value was from 37.66 to 60.30,there were 37 CDS when ENC value＞ 45, and their quantity accounted for 82.22% in all testing genes; there were extremely significant correlation between GC1, GC2, GC3 and GCall, and significant correlation between GC3 and ENC value. The neutrality plot analysis showed that the correlation coefficient of GC12 and GC3 was-0.111 4, the regression coefficient was-0.111 1; ENC-plot analysis revealed that most genes were far from the standard curve;PR2-plot analysis showed that T＞A,G＞C were the base use frequencies of codon in the chloroplast genome of P.kesiya var. langbianensis. These analytic results indicated that the major influencing factors of the codon usage bias in the chloroplast genome P. kesiya var. langbianensis was selection, and mutation might play a smaller role. The present study provided some scientific background for provenance selection and molecular development of P. kesiya var. langbianensis.