Specific primers were designed to amplify the sequences of Hina genes from 121 wild barley (Hordeum spontaneum) accessions belonging to 18 populations from Iran, Israel and Turkey. Forty-nine single nucleotide polymorphisms (SNPs), nine indels, and 26 haplotypes were determined by sequence analysis. The genetic polymorphism (P), genetic diversity (He), and Shannon's information index (I) in the 18 populations were 0.486, 0.181 and 0.269, respectively. Approximately 2/3 genetic variations of Hina genes were presented within populations, while approximately 1/3 genetic variations were observed between populations. Broad gene flow (Nm= 3.31) and low genetic variation (Gst= 0.0702) were detected. However, the genetic differentiation between populations was independent of geographical distances according to the Mantel test (p = 0.478). The result of Spearman rank correlations (rs) showed that the genetic indices (P, He and I) of Hina were not significantly correlated with ecological factors. Only eight SNP positions correlated significantly with ecological factors. Of the eight SNP positions that positively correlated with ecological factors, only one SNP (769, T-C) was located in the coding region; however, it was not responsible for the amino acid change.