Traditional taxonomic classification of the ryegrasses (Lolium sp.) lists eight species. Of these, perennial (L. perenne L.) and Italian (or annual) (L. multiflorum Lam.) ryegrasses have major economic importance as turf and forage in temperate areas of the world. The ryegrass known internationally as annual, L. rigidum Gaud., is a major weed, but can also be used as a forage. Likewise the self-pollinated L. temulentum L. is listed as a noxious weed in 43 U.S. states. Thus, ability to rapidly identify seed of these four species as physical mixtures or originating from pollination among them is a priority for seed testing labs. Generalized RAPD and AFLP marker analyses showed DNA band profiles that differed among the species, but often a distinctive band in bulked samples would not be present in all individuals of one species and the band could be found at a low frequency in other species. The vernalization gene (vrn-1) and genes for the isozymes pgi-2 and sod-1 were cloned from the four ryegrass species based on primers developed from published cereal grain DNA sequences. DNA sequences from the ryegrasses were highly conserved, but polymorphisms were present which appear to be useful for accurately predicting species identity. The results indicate that DNA-based tests for these gene polymorphisms can be further developed to rapidly, inexpensively, and accurately detect contamination of one ryegrass species in seed lots of another.