Cytological and molecular studies were performed on 10 Hordeum species of Iran. The species studied showed the occurrence of 2n=2x=14, 2n=4x=28 and 2n=6x=42. They formed mainly bivalents in metaphase with normal chromosomes segregation during anaphase and telophase stages, except in few cases of laggard chromosomes formation and chromosome stickiness observed in H. bulbosum. H. bulbosum and H. leporinum showed 0–2 B-chromosomes which were smaller than A-chromosomes and did not pair with them out of 20 RAPD primers used 11 primers produced 18 reproducible polymorphic bands. Specific bands were observed in some of the species or populations while, some bands occurred in all the species except one showing the species genetic differences. Clustering of the species based on cytological and RAPD data partly produced similar results, separating H. vulgare and H. spontaneum from each other. The species of H. marinum, H. glaucum and H. leporinum from the sect. Hordeastrum were grouped together, while H. violaceum H. bogdanii and H. brevisubulatum, from the sect. Stenostachys were placed in different clusters separated from each other. H. bulbosum and H. vulgare showed a close relationship, while H. spontaneum, H. distichon and H. vulgare were placed close to each other supporting their taxonomic position in Flora Iranica.