Resistance to biotic stress is one of the most important targets in the improvement of forage and turf grass. Resistance to crown rust in Italian ryegrass is an attractive target of molecular analysis, including linkage analysis and gene isolation, because of its importance in forage and turf grasses. To analyze the major resistance gene in the resistant line ‘Yamaiku 130’, we performed bulked segregant analysis using amplified fragment-length polymorphism (AFLP) in an F1 population segregated at a 1:1 ratio of resistant to susceptible. We constructed a linkage map of regions flanking the resistance gene locus, designated as Pc1. Three AFLP markers were tightly linked to Pc1 with a map distance of 0.9 cM, and 3 AFLP markers were on the opposite side with a distance of 1.8 cM. ATC-CATG153 co-segregated with Pc1. We performed linkage analysis using DNA markers tightly linked to Pc1 in an F1 population derived from the Italian ryegrass cv. ‘Harukaze’. Another resistance gene, designated as Pc2, was identified. Gene isolation of Pc1, using a map-based technique, and the identification of other resistance genes are in progress. Identification of both the DNA markers tightly linked to resistance genes and the plant materials carrying the resistance gene will open new strategies for the development of resistance varieties in Italian ryegrass and related species.