Self-incompatibility (SI) prevents the creation of homozygous parental lines by selfpollination for hybrid breeding in perennial ryegrass (Lolium perenne L.). Although SI is highly effective, its breakdown leading to self-fertility (SF) has been reported. Recent research has fine-mapped one major locus conferring SF in a perennial ryegrass population to a 1.6 cM region on linkage group (LG) 5 (Do Canto et al., 2017). Our goal was to confirm, by marker-trait association using genome-wide markers, this previously described locus as being solely responsible for SF variation.