Abstract Key message We mapped the Rym14 Hb resistance locus to barley yellow mosaic disease in a 2Mbp interval. The co-segregating markers will be instrumental for marker-assisted selection in barley breeding. Abstract Barley yellow mosaic disease is caused by Barley yellow mosaic virus and Barley mild mosaic virus and leads to severe yield losses in barley ( Hordeum vulgare ) in Central Europe and East-Asia. Several resistance loci are used in barley breeding. However, cases of resistance-breaking viral strains are known, raising concerns about the durability of those genes. Rym14 Hb is a dominant major resistance gene on chromosome 6HS, originating from barley’s secondary genepool wild relative Hordeum bulbosum . As such, the resistance mechanism may represent a case of non-host resistance, which could enhance its durability. A susceptible barley variety and a resistant H. bulbosum introgression line were crossed to produce a large F 2 mapping population ( n = 7500), to compensate for a ten-fold reduction in recombination rate compared to intraspecific barley crosses. After high-throughput genotyping, the Rym14 Hb locus was assigned to a 2Mbp telomeric interval on chromosome 6HS. The co-segregating markers developed in this study can be used for marker-assisted introgression of this locus into barley elite germplasm with a minimum of linkage drag.