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Assignment or exclusion of an individual to specific populations or cultivars based on molecular genetic markers provides an attractive approach for varietal identification at the individual level in cross-pollinated plant species. The objectives of this study were (i) to explore the molecular diversity and relationships between Australasian perennial ryegrass (Lolium perenne L.) populations; (ii) to investigate accuracy of assignment of individuals to different types of populations including ecotypic, nonrestricted--and restricted-based cultivars; and (iii) to determine the effect of variable number of SSR loci and different statistical analysis methods on assignment accuracy. Eight forage perennial ryegrass populations comprising 48 individual plants per population were genotyped with 29 simple sequence repeat (SSR) marker loci. The number of alleles per locus ranged from 3.72 to 6.76. The mean observed heterozygosity varied from 0.419 to 0.538. Various genetic distance estimates and clustering methods obtained results consistent with breeding history. Genetic variation among (8.7%) and within populations (91.3%) was significant. Accuracy of individual assignment differed by population type, and it was higher (>90%) for restricted-based cultivars than for ecotypic and nonrestricted-based populations. These results indicate that SSR marker profiles can be effectively used to assign individuals for outbreeding populations such as perennial ryegrass. The approach used in this study may be useful for ryegrass germplasm management issues such as cultivar identification at the individual level.