Abstract Background Qualitative pathogen resistance in both dicotyledenous and monocotyledonous plants has been attributed to the action of resistance (R) genes, including those encoding nucleotide binding site – leucine rich repeat (NBS-LRR) proteins and receptor-like kinase enzymes. This study describes the large-scale isolation and characterisation of candidate R genes from perennial ryegrass. The analysis was based on the availability of an expressed sequence tag (EST) resource and a functionally-integrated bioinformatics database. Results Amplification of R gene sequences was performed using template EST data and information from orthologous candidate using a degenerate consensus PCR approach. A total of 102 unique partial R genes were cloned, sequenced and functionally annotated. Analysis of motif structure and R gene phylogeny demonstrated that Lolium R genes cluster with putative ortholoci, and evolved from common ancestral origins. Single nucleotide polymorphisms (SNPs) predicted through resequencing of amplicons from the parental genotypes of a genetic mapping family were validated, and 26 distinct R gene loci were assigned to multiple genetic maps. Clusters of largely non-related NBS-LRR genes were located at multiple distinct genomic locations and were commonly found in close proximity to previously mapped defence response (DR) genes. A comparative genomics analysis revealed the co-location of several candidate R genes with disease resistance quantitative trait loci (QTLs). Conclusion This study is the most comprehensive analysis to date of qualitative disease resistance candidate genes in perennial ryegrass. SNPs identified within candidate genes provide a valuable resource for mapping in various ryegrass pair cross-derived populations and further germplasm analysis using association genetics. In parallel with the use of specific pathogen virulence races, such resources provide the means to identify gene-for-gene mechanisms for multiple host pathogen-interactions and ultimately to obtain durable field-based resistance.