In this study, 14 wheat cultivars with contrasting yield and N use efficiency (NUE) were used to investigate the agronomic and NUE-related traits, and the N assimilation-associated enzyme activities under low and high N conditions. Under deficient-N, the cultivars with high N uptake efficiency (UpE) and high N utilization efficiency (UtE) exhibited higher plant biomass, yields, and N contents than those with medium and low NUEs. The high UpE cultivars accumulated more N than other NUE type cultivars. Under sufficient-N, the tested cultivars showed similar patterns in biomass, yield, and N content to those under deficient-N, but the varietal variations in above traits were smaller. In addition, the high UpE cultivars displayed much more of root biomass and larger of root length, surface area, and volume than other NUE type cultivars, indicating that the root morphological traits under N deprivation are closely associated with the plant biomass through its improvement of the N acquisition. The high UtE cultivars showed higher activities of nitrate reductase (NR), nitrite reductase (NIR), and glutamine synthetase (GS) at stages of seediling, heading and filling than other NUE type cultivars under both low and high N conditions. Moreover, the high UpE and UtE cultivars also displayed higher photosynthetic rate under deficient-N than the medium and low NUE cultivars. Together, our results indicated that the tested wheat cultivars possess dramatically genetic variations in biomass, yield, and NUE. The root morphological traits and the N assimilation enzymatic acitivities play critical roles in regulating N accumulation and internal N translocation under the N-starvation stress, respectively. They can be used as morphological and biochemical references for evaluation of UpE and UtE in wheat.