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Grain yield and N fertilizer use efficiency (NUE) are closely related to preanthesis root growth and distribution, whereas changes in root morphology and distribution, plants' N uptake and utilization ability, and their relationships with grain yield and NUE remain unclear. We conducted a 2-yr microplot and sand culture experiment using three wheat (Triticum aestivum L.) cultivars (N-efficient, intermediate, and N-inefficient) to examine differences in grain yield, NUE, root growth, root morphology, root distribution, and nitrate transporter gene (TaNRT) expression. The N-efficient wheat cultivar increased grain yield and NUE by improving preanthesis N uptake. Root biomass, growth rate, length, surface area, and weight density were higher in the N-efficient cultivar than in other cultivars at preanthesis. The N-efficient cultivar distributed more roots throughout the topsoil layer. The TaNRT expression level at preanthesis was higher in the N-efficient cultivar than in the intermediate cultivar, whereas no significant genotypic difference was observed after anthesis. This result indicates that TaNRT upregulation increased N efficiency through a higher preanthesis N uptake and that TaNRT 1.1 and TaNRT 1.2 played a more important role than TaNRT 2.1 and TaNRT 2.2. Nitrate reductase and glutamine synthetase activity increased in the N-efficient cultivar at the preanthesis stage and decreased after anthesis. These results indicate that the N-efficient wheat cultivar had higher preanthesis root growth and N uptake ability, promoting leaf N assimilation ability and improving wheat yield and NUE. [ABSTRACT FROM AUTHOR]