The symbiotic association between perennial ryegrass (Lolium perenne L.) and the fungal endophyte Neotyphodium lolii is associated with host-specific adaptations, particularly in response to abiotic and biotic stresses. Knowledge of the origin of the symbiosis and the contribution of endophyte genotype to host phenotypic variation is currently limited. Simple sequence repeat (SSR) markers were used to assess endophyte genetic diversity in a globally distributed collection of perennial ryegrass accessions. Consistent in planta detection was achieved with 18 of 22 SSR markers (primer pairs). Endophytes representing as many as four different taxa were detected in 42 accessions from 20 different countries, N. lolii being predominant. A total of 33 unique N. lolii genotypes were discriminated, of which 29 clustered into three major groups with limited within-group variation. The three major N. loiii groups were associated with distinct perennial ryegrass chloroplast haplotypes. The alkaloid profiles of accessions were apparently associated with the presence of specific N. lolii genotypes. Genotypic analysis provides a powerful method for genetic dissection of the grass-endophyte interaction and prediction of phenotypic variation based on genotypic variation.