||Plant nitrogen assimilation and use efficiency in the seedling’s root system is beneficial for adult plants in field condition for yield enhancement. Identification of genetic basis between root traits and N uptake play a crucial role in wheat breeding. In the present study, 198 doubled haploid lines from the cross of Yangmai 16/Zhongmai 895 were used to identify quantitative trait loci (QTLs) underpinning four seedling biomass traits and five root system architecture (RSA) related traits. The plants were grown under hydroponic cultures with control, low and high N conditions (Ca(NO3)2·4H2O, 0, 0.05 and 2.0 mmol L−1, respectively). Significant variations among the treatments and genotypes, and positive correlations between seedling biomass and RSA traits (r=0.20 to 0.98) were observed. Inclusive composite interval mapping based on a high-density map from the Wheat 660K single nucleotide polymorphisms (SNP) array identified 51 QTLs from three N treatments. Twelve new QTLs detected on chromosomes 1AL (1) in the control, 1DS (2) in high N treatment, 4BL (5) in low and high, 7DS (3) and 7DL (1) in low N treatments, were first reported in influencing the root and biomass related traits for N uptake. The most stable QTLs (RRS.caas-4DS) on chromosome 4DS related to ratio of root to shoot dry weight trait was in close proximity of the Rht-D1 gene, showed high phenotypic effects, explaining 13.1% of the phenotypic variance. Twenty-eight QTLs were clustered in 12 genetic regions. SNP markers tightly linked to two important QTLs clusters C10 and C11 on chromosomes 6BL and 7BL were converted to kompetitive allele-specific PCR (KASP) assays that underpin important traits in root development, including root dry weight, root surface area and shoot dry weight. These QTLs, clusters, and KASP assays can greatly improve efficiency of selection for root traits in wheat breeding programmes.