<p>Diatoms, as the important primary producers in the ocean, play a crucial role in the marine carbon cycle. Despite their crucial role in marine biogeochemical cycles and carbon sequestration, detailed analyses of codon usage patterns in diatom chloroplast genomes have been limited. This study investigates the codon usage bias (CUB) in the chloroplast genomes of 12 representative diatoms within the family <i>Thalassiosiraceae</i> and <i>Skeletonemataceae</i>. In the present research, codon composition and factors influencing CUB were analyzed. The comprehensive research into the proliferation of the chloroplast genome unveils a distinct preference for A and T(U) in the codon usage of diatom chloroplast genomes, with significant variations in GC content across different codon positions. Our results also reveal that the chloroplast genes of these diatoms exhibit specific CUB, with some codons being preferentially utilized over others. Meanwhile, six optimal codons are shared among the chloroplast genomes of 12 diatom species: UUU, GGA, CGA, AAU, CAU, and UAU. Based on the effective number of codons (ENC) and relative synonymous codon usage (RSCU) results, we propose that natural selection is the predominant mechanism contributing to the CUB. Moreover, the Ka/Ks ratios of 265 orthologous coding genes also indicate that these chloroplast genomes were predominantly subjected to purifying selection during the evolutionary process. Our study reveals the pattern of CUB in diatoms and provides valuable information for future genetic engineering of microalgae. These findings may also inform strategies for enhancing CO₂ fixation by diatoms and offer perspectives on microalgal adaptation to environmental changes.</p>