Phylogenetic relationships within the genus Hordeum were investigated based on nucleotide sequences of the thioredoxin-like (HTL) gene. We analyzed amplified genomic DNA fragments of the HTL gene from 11 Hordeum species including 16 taxa (25 accessions), which cover mainly diploid accessions together with several tetraploid accessions. Phylogenetic analysis based on the HTL sequences demonstrated a clear divergence of the four basic genomes I (H. vulgare and H. bulbosum), Xa (H. marinum), Xu (H. murinum) and H (other species) in the genus. Phylogenetic clustering also led us to infer two separate clades, one containing the I and Xu, and the other containing the Xa and H genomes. In the diploid H-genome species, American species were confirmed to be closely related and divergent from Asian species. Analysis of the Xa-genome group suggested an alloploid origin of tetraploid H. marinum ssp. gussoneanum by hybridization between the diploid cytotypes of ssp. marinum and ssp. gussoneanum. Our data also supported the hypothesis that two tetraploid cytotypes of H. murinum (ssp. murinum and ssp. leporinum) have an alloploid origin, where one genome was presumably derived from the diploid cytotype ssp. glaucum and the other from an unknown Xu-genome species.