Abstract: Four representatives of wild relative and cultivar of barley, i. e. two rowed wild barley Hordeum vulgare L. ssp. spontaneum (Koch) Hsü, six rowed wild barley H.vulgare L.ssp.agriocrithon (Åberg) Hsü, six rowed bottle shaped wild barley H.vulgare L. ssp. agriocrithon var. lagunculiform (Bakht) Hsü and cultivated barley H. vulgare L. ssp. vulgare Hsü from Qing Zang Plateau were studied by in situ hybridization using 5S rRNAgenes as probe.Located 5S rRNAgenes sites were used to construct an idiogram, which provided as molecular karyotype.It showed that 5S rRNAgenes in barley were movable taking into account of its localization and, total number of sites for 5S rRNAgenes increased from two rowed wild barley, six rowed bottle shaped wild barley to six rowed wild barley and cultiva ted barley.The relationship between evolution of 5S rRNAgenes and that of barley as well as the application of in situ hybridization in phylogeny were discussed.