Ten chloroplast microsatellite markers were used to characterise chloroplast genetic diversity at allelic and haplotypic level in 104 accessions of Lolium perenne, other Lolium species, Festuca species and x Festulolium cultivars. Furthermore, genetic relationships between the accessions and biogeographic distribution of haplotypes were investigated using a range of Nei's population genetic diversity measures and analysis of molecular variance (AMOVA). An extremely high number (511) of haplotypes was detected in 1575 individuals. Nei's gene diversity values among L. perenne accessions ranged between 0 and 0.333. Much of the L. perenne European ecotype diversity (61%), as calculated using AMOVA, could be attributed to within-population variance and this is likely caused by, and maintained by, high levels of natural and anthropogenic seed dispersal. Plastid gene pools and maternal lineages for L. perenne could be clearly identified. Evidence was found, using AMOVA, to show a likely migration route of L. perenne from Southern regions of Europe northwards.