Key message <p>Field phenotyping of a recently established NAM population for stem rust resistance, combined with high-density genotyping, enabled the identification of novel sources of stem rust resistance in Italian ryegrass.</p> Abstract <p>Stem rust, caused by <i>Puccinia graminis</i> ssp. <i>graminicola</i>, is a major disease affecting the outcrossing species Italian ryegrass (<i>Lolium multiflorum</i> Lam.) and leading to substantial reductions in seed yield. Until now, knowledge on the genetic control of stem rust resistance in Italian ryegrass has been limited to a few quantitative trait loci identified in biparental mapping populations. To discover novel resistance sources for use in breeding programs, appropriate plant populations, reliable phenotyping methods and advanced genomic tools are essential. In this study, we utilized a previously established <i>F</i>₂ nested association mapping (NAM) population comprising 708 individuals derived from 24 founder plants, exhibiting high variation in stem rust resistance. Phenotypic evaluation was conducted under natural inoculation in three location-by-year combinations. By integrating reduced-representation sequencing of the NAM population with whole genome sequencing of the founder plants, we identified 3,199,253 SNP markers for association mapping. The high SNP marker density, together with the strong detection power of the NAM population, enabled the identification of four novel candidate genes. Two of these genes, located on chromosomes 6 and 7, encode receptor-like serine/threonine kinases that are known to play a role in stem rust resistance in other crops. Within the serine/threonine kinase gene <i>Chr7.32208</i>, two superior haplotypes were identified that can be directly implemented as selection criteria in breeding programs. The novel stem rust resistance candidate genes reported here provide promising targets for functional validation and the improvement of stem rust resistance in Italian ryegrass breeding.</p>