The 18S-5.8S-26S rDNA and 5S rDNA loci have been mapped physically by fluorescent in-situ hybridization to the chromosomes of Phalaris coerulescens. The biotin-labelled heterologous 18S-5.8S-26S rRNA probe (pTa71) detected one locus, which corresponded to the secondary constriction (nucleolar organizer) on the long arm of the satellited chromosome II. The homologous 5S rDNA probe (Bam2.12) detected two pairs of 5S rRNA gene clusters which were localized at two different non-satellited chromosomes, one near the telomere on the short arm of the chromosome I, which is the largest chromosome of the complement, and the other about 42% out on the long arm of the chromosome III. A BamHI fragment, containing the 5S rRNA gene, has been isolated and characterized. The 5S rDNA repeat unit is 309 bp in length, consisting of 121 bp highly conserved coding region and 188 bp variable spacer region. The karyotype or Phalaris cocrulescens is characterized by the similar size of chromosomes within the group 2, group 3, or group 4. This study represents the first step towards the understanding the genome organization of Phalaris coerulescens and provides reliable markers for chromosome identification in this grass, an important species as a model system for the study of self-incompatibility in grasses.