Breeding wheat cultivars with high nitrogen use efficiency is crucial for sustainable agriculture. In this study, 130 durum wheat accessions from 53 countries were cultivated under two nitrogen fertilization levels to investigate variations in agronomic traits and identify accessions suitable for low-nitrogen conditions. Under no nitrogen fertilization, SPAD value (-99.2), flag leaf length (−15.8 mm), grain area (−0.52 mm²), grain length (−0.27 mm), thousand-grain weight (+5.2 g), and grain protein content (−4.2%) showed significant differences compared to that under standard nitrogen fertilization. Among these traits, the SPAD value exhibited a strong positive correlation with protein content in the absence of nitrogen fertilization, suggesting its potential as an indicator for selecting germplasm with superior nitrogen use efficiency. Hierarchical cluster analysis, based on the differences in the six traits under the two nitrogen fertilization levels, classified the 130 durum wheat accessions into three groups (Groups 1, 2, and 3). Accessions in Group 2 exhibited superior adaptability under no nitrogen fertilization compared with those in the other two groups. We identified seven accessions in Group 2 that exhibited minimal decreases in SPAD values and protein content under no nitrogen fertilization for use in wheat breeding programs aimed at developing cultivars with high nitrogen use efficiencies.