Abstract: In this study, we sequenced the chloroplast genome of Quercus austroglauca Y. T. Chang and analyzed the structural characteristics of the chloroplast genome and factors influencing codon preference using various software. Results showed that the chloroplast genome of Q. austroglauca was 160 913 bp in length, encoding 133 genes. Analysis of 52 screened protein-coding sequences revealed an average GC content of the third base of 29.14%, signifying a pronounced preference for A or U as the terminal codon. These results were confirmed by relative synonymous codon usage analysis. Number of effective codons (ENC) was greater than 35, indicating weak codon preference. Neutral mapping, ENC-plot, and PR2-plot analyses showed that natural selection was the dominant factor affecting codon preference in the Q. austroglauca chloroplast genome. In total, 13 optimal codons were identified by screening. Multiple analysis showed that variation mostly occurred in the non-coding regions, although the ycf1 gene exhibited a large degree of variation in the coding region. Inverted repeat (IR) boundary analysis identified a ycf1 pseudogene in Q. austroglauca Phylogenetic analysis showed that Q. austroglauca was closely related to Q. hypargyrea (Seemen) C. C. Huang et Y. T. Chang.