Imbalanced use (form, quantity, and ratio) of nitrogen fertilization can result in decreased grain yields and increased nitrogen loss, leading to adverse effects on overall environmental quality. Globally, limited empirical research has been conducted on the comprehensive effects of different levels of N that can significantly influence wheat agronomic and genotypic traits. Therefore, this study aimed to evaluate wheat genotypes for two consecutive years (2020–2021 and 2021–2022) under different N fertilization treatments: N0 (native N, without external application of N), N75 (½ of the recommended dose of N), and N150 (recommended dose of N). The study findings revealed that ‘HD 3249’ and ‘HD 3117’ were the top-performing genotypes in terms of grain yield (5.3 t ha−1; 5.0 t ha−1), straw yield (6.9 t ha−1; 6.7 t ha−1), biological yield (12.2 t ha−1; 11.8 t ha−1), and harvest index (42.9%; 42.4%). In particular, the application of N75 and N150 increased grain yields by 142.6% and 61.3%, respectively; straw yields by 72.3%; and by 110.6% over N0. Furthermore, N levels (N75 and 150) significantly increased the higher concentration of N in grain (23.1% and 33%) and straw (21.1% and 29.8%); N uptake in grain (70.2 and 104.2) and straw (64.8 and 41.5); and total N uptake (68.8% and 101.4%) than N0, respectively. Additionally, correlation analysis revealed that there were positive correlations between yields, harvest index as well as N concentration and uptake. This study identified the two elite genotypes, ‘HD 3249’ and ‘HD 3117’, with N150 splits giving a better response, which can be used as selection criteria for developing wheat varieties that are more efficient in using nitrogen, leading to high yields and N uptake.