Abstract The genusTrifolium(Leguminosae), which includes several important forage legumes of temperate and subtropical regions, is characterized by small chromosomes of uniform size. Cytogenetic mapping of the two multigene families coding for 18S-5.8S-26S rRNA and 5S rRNA in white clover (T. repens)and seven closely related species/subspecies was carried out using fluorescencein situhybridization (FISH). The 18S-26S rDNA, generally confined to nucleolus organizer regions (NORs), was consistently located proximally on the shorter arm of one or two metacentric or submetacentric chromosome pairs. DAPI-negative, diffused and highly stretched 18S-26S FISH signals, representing previously transcriptionally active NORs, were observed up to late metaphase. The flanking condensed ends of the diffused 18S-26S rDNA signals were DAPI-positive and represented perinucleolar knobs which are presumed to be transcriptionally inactive. In interphase cells, the decondensed NOR chromatin passed through the nucleolar domain. In one species (T. isthmocarpum),in addition to two NOR chromosome pairs, 18S-26S signals were also located on a third pair, with no apparent NOR association. The 5S rDNA signals were located proximally on a long arm in six species and were syntenic to the NORs in all these species exceptT. ambiguumandT. nigrescensssp.petrisavii.In two species (T. hybridum, T. isthmocarpum),5S sequences occurred in the chromosomal satellites aligned distally to the NOR sequences. The relative distribution patterns of the two types of tandemly repeated DNA sequences were species-specific except forT. nigrescensssp.petrisaviiandT. ambiguum,both of which showed identical hybridization patterns. The two types of rDNA sequences have provided molecular markers for individual identification of a set of clover chromosomes. Present findings support the allotetraploid origin of white clover (T. repens)and suggest thatT. nigrescensssp.petrisaviimay be one of the present day diploid ancestors.