To date, little molecular genetic research has been conducted in Lolium and the current study is the first investigation of diversity in ribosomal DNA (rDNA) among perennial ryegrass cultivars. Ribosomal DNA probes derived from the intergenic spacer (IGS–M) and transcribed (COD–M) regions of mustard (Sinapis alba) were used to examine 35 perennial ryegrass (L. perenne L.) cultivars for genetic diversity using RFLP methodology. Analysis of digested genomic DNA on Southern blots revealed only 10 cultivars with unique RFLP patterns. Genetic diversity was exclusively associated with the IGS–M probe, as the cultivars were all indistinguishable when examined with the COD–M probe. This low incidence of distinctions was not expected, given that the 35 cultivars were known genetically distinct entities, representing the very wide range of the major phenotypic and physiological characteristics present in commercial stocks. Furthermore, the forage cultivars were proportionately less polymorphic in comparison to the amenity cultivars. This was also unexpected as amenity cultivars are recognized as a more closely clustered group than the forage cultivars, based on morphological characteristics. The potential use of rDNA for assessing cultivar diversity and breeding histories in perennial ryegrass is considered.