Against a backdrop of a changing climate and population, new strategies are required to breed plants with improved productivity under different environments and stress conditions. In the past, breeding for enhanced crop yield has been focusing on improvement of the aerial parts of plants, which today incorporates the implementation of non-invasive highthroughput phenotyping approaches. In contrast, the plant roots are largely ignored in most crops, although roots are essential for yield and adaptation of crops to biotic and abiotic stress. Direct phenotyping of root traits is laborious and difficult, and implementation of root phenotyping approaches into breeding programmes has been a major challenge in the past. Thus, implementation of marker-assisted selection for root traits is a classical example where plant breeders could strongly benefit from development of easily manageable, high-throughput SNP genotyping marker assays for selection of root traits. The intention of this study was to develop breeder-friendly Kompetitive Allele Specific PCR (KASP) markers for marker-assisted backcrossing of a QTL conferring high root dry biomass (RDM) in wheat. Using 90k iSelect wheat SNP array genotyping data, this major-effect QTL could be associated with two additive haplotype blocks (Hap-5B-RDMa and Hap-5BRDMb) on chromosome 5B in a global bread wheat panel. We attempted to convert 15 SNP markers spanning these haplotype blocks into KASP markers. All 6 SNPs located in haploblock Hap-5B-RDMb were successfully converted into locusspecific KASP markers. In contrast, only 3out of 9 SNPs located in haploblock Hap-5B-RDMa could be converted into locusspecific KASP markers. Limited transferability to locus-specific KASP primers for haploblock Hap-5B-RDMa was caused by high sequence similarity with corresponding regions on chromosomes 5A and 5D and insufficient coverage of haploblock Hap-5B-RDMa diversity by the wheat reference genome. However, 3 reproducible, stable KASP assays could be designed which are sufficient to differentiate between accessions with high and low root biomass. The new markers are being used for marker-assisted backcrossing. The results provide a basis to set general guidelines for successful KASP marker development in wheat and other polyploid crops.