Several linkage maps, mainly based on anonymous markers, are now available for Lolium perenne. The saturation of these maps with markers derived from expressed sequences would provide information useful for QTL mapping and map alignment. Therefore, we initiated a study to develop and map DNA markers in genes related to self-incompatibility, disease resistance, and quality traits such as digestibility and sugar content in two L. perenne families. In total, 483 and 504 primer pairs were designed and used to screen the ILGI and CLO-DvP mapping populations, respectively, for length polymorphisms. Finally, we were able to map 67 EST markers in at least one mapping population. Several of these markers coincide with previously reported QTL regions for the traits considered or are located in the neighbourhood of the self-incompatibility loci, S and Z. The markers developed expand the set of gene-derived markers available for genetic mapping in ryegrasses.