Crown rust resistance is an important selection criterion in ryegrass breeding. The fungal disease caused by P. coronata causes yield loss and a reduced quality of the fodder crop. Molecular markers were used to unravel the genomic organization of crown rust resistance in a segregating L. perenne population. Two genomic regions involved in crown rust resistance were identified that together explained 35% of the phenotypic variance present. Bulked segregant analysis in combination with AFLP markers was a suitable method to identify DNA markers associated with genomic regions of major effect. One cluster of AFLP markers explained 6.1% of the variance and mapped to linkage group 2, a genomic region known to contain crown rust resistance genes. A second cluster of AFLP markers detected a novel genomic region of major effect that explained 27.7% of the phenotypic variance in crown rust resistance. This cluster was unlinked to the cluster on linkage group 2. Divergent selections performed within the segregating F-1 population on the basis of genotype and phenotype revealed that the markers associated with crown rust resistance identified in this study have potential for marker assisted selection. Selection of plants on the basis of markers was more straightforward than the selection on the basis of phenotype.