This study assessed the genetic diversity in 928 individuals from 40 diploid populations of Lolium perenne using nuclear simple sequence repeat markers, including 22 accessions of Irish ecotypes, seven European ecotypes and 11 released varieties. High levels of allelic and genetic diversity were determined, with intra-population variation accounting for the majority of the variation. The majority of the accessions deviated from Hardy–Weinberg equilibrium and had relatively high inbreeding coefficients. Two major gene pools of ecotypic accessions were defined by unweighted pair group method with arithmetic mean (UPGMA) and PCA analyses. One of these two gene pools accounted for two-thirds of the ecotypes and included most of the current Irish and Northern Irish breeding materials and about half of the European ecotypes included in this study; these European ecotypes performed well under Irish selection conditions. Population structure and differentiation analyses using Structure analysis and analysis of molecular variance confirmed the results found in the UPGMA and PCA analyses. These results will be useful for breeders who wish to exploit specific pools from ecotype collections.