The market success of perennial ryegrass (Lolium perenne L.) cultivars depends on sufficient seed production, as they are propagated by seed. However, breeding for high quality forage production reduces seed yield, and breaking the negative correlation would help to overcome the problem. The foliar disease crown rust is another factor affecting reproductive capacity and thereby seed yield. We evaluated seed yield-related traits and resistance to crown rust in a collection of commercial cultivars and ecotypes of perennial ryegrass and identified genome-wide markers associated with the traits. The study revealed high variation between the ecotype and cultivar groups as well as between years. A genome-wide association study identified 17 DNA single-nucleotide polymorphisms (SNPs) of which eight were associated with crown rust and nine with flag-leaf length. The SNP markers were located within or near predicted genes functioning in defense against pathogens. The identified genes are strong candidates for a further in-depth functional study to continue unravel determination of leaf architecture and crown rust resistance in perennial ryegrass.