The 18S-26S ribosomal genes in three closely related species of Leymus (Poaceae: Triticeae) were examined using fluorescence in situ hybridization (FISH) and restriction fragment length polymorphism (RFLP). Both approaches revealed a close relationship between L. arenarius (8x = 56, northern European) and L. racemosus (4x = 28, central Eurasian), whereas L. mollis (4x = 28, northern American/Pacific) was distinct. Each species had three homologous pairs of major rDNA loci: a1, a2, and a3 for L. arenarius; m1, m2, and m3 for L. mollis; and r1, r2, and r3 for L. racemosus. Leymus arenarius had in addition three minor loci, a4, a5, and a6. The major loci of L. arenarius and L. racemosus were identical, indicating that the former species could have originated from the latter, via interspecific hybridization and/or polyploidy. The rDNA-RFLPs further indicated relationships of these species to other species of Leymus (L. karellini, 8x = 56 and L. angustus, 12x = 84) and Psathyrostachys (P. fragilis, P. huashanica, P. juncea, and P. lanuginosa, which are all diploids). A phenogram constructed from 20 BamHI, EcoRI, and DraI rDNA fragments revealed closer relationship between the two genera, Leymus and Psathyrostachys, than that among species within a genus.