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Isozyme electrophoresis was carried out on 423 accessions belonging to four species of the genus Lolium to assess the genetic variation within and between the species. Four enzyme systems (acid phosphatase, glutamate oxaloacetate transaminase, phospho-gluco isomerase and superoxide dismutase) were used to array allelic diversity at five polymorphic loci. Nei's genetic distance analysis and multivariate analyses were computed and the taxonomic position of each species is discussed as a result of these computations. Electrophoresis is shown to clearly differentiate the inbreeding species, L. temulentum from the outcrossing species, L. perenne, L. multiflorum and L. rigidum, and to separate the species within the out-breeding group. The taxonomic results are discussed in relation to an earlier paper on morphological variation, concluding that electrophoresis is a valid taxonomic tool showing distinct separations between the species, but that current plant breeding and agricultural practises may be increasing the amount of hybridisation between the species than occurred in the past.