The Hina gene is one of the two known Hin genes for hardness, and its RNA expression is correlated with grain hardness and dry matter digestibility variation. In this study, only one clone of Hina gene was obtained from one barley accession. A total of 121 Hina gene sequences were isolated from 121 wild barley (Hordeum spontaneum) accessions in Israel, Iran, and Turkey, and then their molecular characteristics were compared with 97 Hina gene sequences from 74 cultivated barley (H. vulgare) lines in Europe and 23 landrace (H. vulgare) with global distribution and other 26 Hina gene sequences from cultivated barleys (H. vulgare) with unknown global distribution. Cis-acting regulatory element (CARE) searching revealed that there were different types of regulatory element for the Hina gene in wild and landrace/cultivated barleys. There were six consistent cis-acting binding sites in wild and landrace/cultivated barleys, whereas 8 to 16 inconsistent TATA-boxes were observed. In addition, three special elements (E2Fb, Sp1, and boxS) were only observed in wild barley, while one (AT1-motif) was only found in landrace/cultivated barley. Forty-four deduced amino acid sequences of HINA from wild and landrace/cultivated barleys were obtained by deleting repetitive amino acid sequences, and they were clustered into two groups on the basis of Neighbor-Joining analysis. However, there was no obvious difference in the amino acid sequences of HINA between wild and landrace/cultivated barleys. Comparing to protein secondary structure of wheat PINA, it was indicated that HINA also existed a signal peptide. In addition, HINA was a hydrophilic protein on the basis of the protein properties and composition. The Hina gene is one of the two known Hin genes for hardness, and its RNA expression is correlated with grain hardness and dry matter digestibility variation. In this study, only one clone of Hina gene was obtained from one barley accession. A total of 121 Hina gene sequences were isolated from 121 wild barley (Hordeum spontaneum) accessions in Israel, Iran, and Turkey, and then their molecular characteristics were compared with 97 Hina gene sequences from 74 cultivated barley (H. vulgare) lines in Europe and 23 landrace ( H. vulgare) with global distribution and other 26 Hina gene sequences from cultivated barleys (H. vulgare) with unknown global distribution. Cis-acting regulatory element (CARE) searching revealed that there were different types of regulatory element for the Hina gene in wild There were six consistent cis-acting binding sites in wild and landrace / cultivated barleys, but 8 to 16 inconsistent TATA-boxes were observed. In addition, th ree special elements (E2Fb, Sp1, and boxS) were only observed in wild barley, while one (AT1-motif) was only found in landrace / cultivated barley. Forty-four deduced amino acid sequences of HINA from wild and landrace / cultivated barleys However, there was no obvious difference in the amino acid sequences of HINA between wild and landrace / cultivated barleys. Comparing to protein secondary structure of wheat PINA, it was indicated that HINA also existed a signal peptide. In addition, HINA was a hydrophilic protein on the basis of the protein properties and composition.