Wild species in the Triticeae tribe are very valuable resources for agronomic improvement in cereal crop species. Intergeneric hybrids were produced between several barley cultivars and perennial species in the genera Elymus, Thinopyrum, and Pseudoroegneria. Caryopsis formation and subsequent plantlet regeneration from embryo culture were variable depending on the hybrid combinations. Chromosome numbers and hybrid identity were confirmed by GISH analysis on the somatic cells of the hybrids. While the hybrids showed very robust vegetative growth and exceeded the parental spikes in size, their floral morphologies resembled that of the wild species. Meiotic chromosome analysis revealed that the bivalent formation frequency per cell ranged from 0.06 in Hordeum vulgare 'Betzes' x Elymus curvatus to 3.0 in Elymus humidus x H. vulgare 'Manley'. By GISH analysis on the meiocytes of the hybrid E. humidus x 'Manley', the frequency of autosyndetic bivalents exceeded the allosyndetic bivalent formation, which gave an insight into the genome constitution of E. humidus as an autoallohexploid species. Regardless of the low allosyndetic chromosome pairing between barley and E. humidus, this combination may be useful for further input, since E. humidus is known to carry many valuable genes for biotic and abiotic stress tolerance.