Background and aims: Deep roots are needed to allow uptake of nitrogen (N) and water available in the deeper soil layers, to help tolerate increasingly extreme climates. Yet few studies in the field have been able to identify genetic differences in deep roots and how this relates to N and water uptake. This study aimed to identify the relationship between deep roots and tolerance to drought, and how this varies by genotype and with differing N fertilization. Methods: We grew 14 diverse genotypes of winter wheat in a semi-field facility in Denmark, in 2019 and 2020, with a soil depth gradient and a rain-out shelter to create a water stress. We used minirhizotron tubes reaching to 2.5 m depth to quantify differences in deep roots. We applied isotope tracers (¹⁵N and ²H labelled water) at 1.6-1.8 m at anthesis to assess differences in root function. Grain and straw ¹³C were used to assess drought stress. Results: We found differences in deep roots between genotypes, and slightly less deep root growth when more N was applied. Deep roots were correlated with grain yield, uptake of deep-placed tracers of water and N, and tolerance to drought. Genotypes with deeper roots had the biggest decrease in water stress and increase in grain yield, when their roots had access to deeper soil. Conclusion: Deeper roots were related to drought tolerance and increased yields. This suggests that deep rooting should be considered in future breeding efforts for more climate resilient crops.