A mapping population was created to detect quantitative trait loci (QTL) for resistance to stem rust caused by Puccinia graminis subsp. graminicola in Lolium perenne. A susceptible and a resistant plant were crossed to produce a pseudo-testcross population of 193 F(1) individuals. Markers were produced by the restriction-site associated DNA (RAD) process, which uses massively parallel and multiplexed sequencing of reduced-representation libraries. Additional simple sequence repeat (SSR) and sequence-tagged site (STS) markers were combined with the RAD markers to produce maps for the female (738 cM) and male (721 cM) parents. Stem rust phenotypes (number of pustules per plant) were determined in replicated greenhouse trials by inoculation with a field-collected, genetically heterogeneous population of urediniospores. The F(1) progeny displayed continuous distribution of phenotypes and transgressive segregation. We detected three resistance QTL. The most prominent QTL (qLpPg1) is located near 41 cM on linkage group (LG) 7 with a 2-LOD interval of 8 cM, and accounts for 30-38% of the stem rust phenotypic variance. QTL were detected also on LG1 (qLpPg2) and LG6 (qLpPg3), each accounting for approximately 10% of phenotypic variance. Alleles of loci closely linked to these QTL originated from the resistant parent for qLpPg1 and from both parents for qLpPg2 and qLpPg3. Observed quantitative nature of the resistance may be due to partial-resistance effects against all pathogen genotypes, or qualitative effects completely preventing infection by only some genotypes in the genetically mixed inoculum. RAD markers facilitated rapid construction of new genetic maps in this outcrossing species and will enable development of sequence-based markers linked to stem rust resistance in L. perenne.