The genetic diversity and relationships of 48 Lolium temulentum accessions derived from eight countries (Morocco, Egypt, Tunisia, Italy, Ethiopia, Pakistan, Nepal and Japan) were analysed using seven microsatellite and 44 AFLP polymorphic loci to investigate the origin and distribution of genetic variation. Cluster analysis was performed using the unweighted pair-group method with arithmetic averages (UPGMA) based on the simple matching coefficient of similarity. Nei's gene diversity (h) and the coefficient of gene differentiation (Gst) were calculated. The results from microsatellite analysis indicated that accessions from the same country did not always cluster together, probably because of the limited number of loci used. In contrast, AFLP clearly separated clusters between countries and/or regions; Pakistan–Nepal complex, the Mediterranean region, Ethiopia and Japan. The h value was much higher for microsatellite than for AFLP analysis, indicating that microsatellites are the more variable markers. For AFLP analyses, h values were highest in accessions from Pakistan–Nepal complex, and from the Mediterranean region. These are in agreement with proposals that the origin of L. temulentum lies between the South-western Asia and the Mediterranean basin. The clear groupings of accessions from each country and/or region with high Gst (0.688) indicate that exchange of seeds between them is limited.