Abstract: Hordeum agriocrithon is considered as the wild progenitor of six-rowed cultivated barley and is widely distributed in the Qinghai-Tibet Plateau. In order to reveal the structural characteristics and phylogenetic relationship of chloroplast DNA（cpDNA） of H. agriocrithon, the H. agriocrithon cpDNA was sequenced by using the Illumina Hiseq 6000 sequencing platform, and the cpDNA of H. spontaneum was used as a reference to sequence assembly and quality control. The cpDNA of H. agriocrithon was annotated and characterized by bioinformatics. Meanwhile, the maximum likelihood method（ML） phylogenetic tree was constructed by MEGA X software, with Zea mays and Gramineae Stipa species Stipa lipskyi as outgroups, to analyze the phylogenetic relationships of Hordeum agriocrithon. The results showed that the cpDNA of H. agriocrithon was 136 462 bp in length, with a G+C content of 38.32%, which was a typical tetrad structure, including a pair of inverted repeats region（IRs） of 21 582 bp, a large single copy region（LSC） of 80 597 bp, and a small single copy region（SSC） of 12 701 bp. A total of 129 genes were identified in the cpDNA of H. agriocrithon, including 83 protein-coding genes, 38 tRNA genes, and 8 rRNA genes. A total of 46 dispersed repeated sequences and 178 simple sequence repeats（SSR） loci were detected, most of SSR were single base repeats composed of A/T. Codon usage bias analysis showed that H. agriocrithon had a preference and preferred to use the codons ending with A/U. IR boundary analysis combined with comparative sequence analysis showed that the chloroplast genome structures of H. agriocrithon, H. distichon, H. spontaneum, and H. vulgare were highly conserved, in which the sequence variation mainly occurred in the non-coding sequence of the LSC, except for H. marinum. The phylogenetic analysis showed that the H. agriocrithon clustered with Hordeum species and had the closest genetic relationship with H. vulgare. In this study, the complete chloroplast genome sequence of H. agriocrithon was obtained, which clarified the affinities between the Triticeae and provided the scientific basis for the study of the phylogeny and species identification of H. agriocrithon.