Sixteen wild populations of Lolium rigidum Gaud. were collected from island and mainland locations in Mediterranean Europe and western Asia. Five loci, controlling isozymes at four enzyme systems, were assayed on 60 plants per population using starch gel electrophoresis. Gene diversity measures and Nei’s genetic distance were calculated, and the relationships between the populations were analysed using principal component (PCA) and cluster analyses. All populations were found to show a gene diversity index that is consistent with that expected in a cross-fertilising species. The highest gene diversity measures were recorded in some of the island populations. Both PCA and cluster analysis grouped the Balearic Island populations together and separated the Spanish and Sardinian populations from any of the others. The results are discussed with respect to a number of theories of migration between populations, and in relation to the colonization history of each island. It is suggested that the preservation of these island populations in gene banks is important as part of a core collection of the species.