Italian ryegrass (Lolium multiflorum Lam.) is one of the most important forage grass species in temperate regions. Its yield, quality and persistency can significantly be reduced by bacterial wilt, a serious disease caused by Xanthomonas translucens pv. graminis. Although a major QTL for bacterial wilt resistance has previously been reported, detailed knowledge on underlying genes and DNA markers to allow for efficient resistance breeding strategies is currently not available. We used pooled DNA sequencing to characterize a major QTL for bacterial wilt resistance of Italian ryegrass and to develop inexpensive sequence-based markers to efficiently target resistance alleles for marker-assisted recurrent selection. From the mapping population segregating for the QTL, DNA of 44 of the most resistant and 44 of the most susceptible F1individuals was pooled and sequenced using the Illumina HiSeq 2000 platform. Allele frequencies of 18 × 106single nucleotide polymorphisms (SNP) were determined in the resistant and susceptible pool. A total of 271 SNPs on 140 scaffold sequences of the reference parental genome showed significantly different allele frequencies in both pools. We converted 44 selected SNPs to KASP™ markers, genetically mapped these proximal to the major QTL and thus validated their association with bacterial wilt resistance. This study highlights the power of pooled DNA sequencing to efficiently target binary traits in biparental mapping populations. It delivers genome sequence data, SNP markers and potential candidate genes which will allow to implement marker-assisted strategies to fix bacterial wilt resistance in outcrossing breeding populations of Italian ryegrass.