Codon usage bias, which exists in many genomes, is mainly determined by mutation and selection. To elucidate the genetic features and evolutionary history of herbaceous peony (Paeonia lactiflora), a well-known symbol of prosperity in China, we examined synonymous codon usage in 24,216 reconstructed genes from the P. lactiflora transcriptome. The mean GC content was 44.4%, indicating that the nucleotide content of P. lactiflora genes is slightly AT rich and GC poor. The P. lactiflora genome has a wide range of GC3 (GC content at the third synonymous codon position) distribution, with a significant correlation between GC12 and GC3. ENC (effective number of codons) analysis suggested that mutational bias played a major role in shaping codon usage. Parity Rule 2 (PR2) analysis revealed that GC and AU were not used proportionally. We identified 22 “optimal codons”, most ending with an A or U. Our results suggested that nucleotide composition mutation bias and translational selection were the main driving factors of codon usage bias in P. lactiflora. These results lay the foundation for exploring the evolutionary mechanisms and heterologous expression of functionally-important proteins in P. lactiflora.