The codon usage preferences and differences among cultivated and wild barley， Hordeum vulgare var. Coleste， H. vulgare ssp. vulgare and H. vulgare ssp. spontaneum， were analyzed based on the coding sequences of their chloroplast genomes. The results demonstrated that the cytosine and guanine （GC） content at different codon positions （GC1， GC2， GC3） exhibited a gradient decrease across the three species （46.74%/46.80%/46.63%， 39.47%/39.43%/39.43%， 29.80%/29.75%/30.25%）. All three species shared 31 highly preferred codons （RSCU > 1）， with 29 of these codons ending in A/U， indicating a significant preference for NNA/NNU endings. The effective codon number （ENC） values of the chloroplast genomes （47.14， 47.02， 47.75） and the proportion of genes with ENC > 45 （39， 39， 42） suggested a relatively weak overall codon preference. Neutral plotting， ENC-plot， and PR2-plot analyses confirmed that natural selection was the primary driving force behind the formation of codon preferences. Furthermore， two cultivated barley exhibited high convergence in terms of GC composition， ENC distribution， and optimal codons （including specific GCA/AGA）. This suggests that artificial selection through purifying selection has enhanced the genetic stability of beneficial traits， offering new insights into the molecular mechanisms underlying barley domestication.