Background: Large quantities of nitrogen (N) fertilizer applied to cotton cropping systems support high yields but cause adverse environmental impacts such as N 2 O emission and water eutrophication. The development of cotton cultivars with higher N use efficiencies suitable for low-N conditions is therefore important for sustainable production. In this study, we evaluated 100 cotton genotypes in 2016 for N use efficiency and related traits at the seedling stage. Methods: Sand culture experiment was conducted with low N levels (0.01 g•kg − 1) or normal N levels (0.1 g•kg − 1). We investigated plant height, SPAD value (soil plant analysis development chlorophyll meter), dry weight, N accumulation, N utilization efficiency, and N uptake efficiency. Through descriptive statistics, principal component analysis and heatmap clustering analysis, we confirmed the evaluation index system of N-efficient genotypes and the classification of N-efficient genotypes. Results: Significant differences were observed among N levels and genotypes for all agronomic traits and N levels. Coefficients of variation varied greatly and ranged from 6.7~28.8 and 7.4~20.8 under low-N and normal-N treatment, respectively. All traits showed highly significant positive correlations with each other, except SPAD value. The principal components under both N levels were similar, showing that total dry weight, aboveground dry weight, total N accumulation, and N uptake efficiency were important components. We confirmed these four traits as suitable screening indexes for low N tolerance. Based on the results of heatmap clustering and scatter diagram analysis of N efficiency value, 10 genotypes were found low-N tolerant, in which five varieties were inefficient under both low and normal N conditions, while four varieties were found efficient under low-N conditions but inefficient under normal-N conditions. Only one variety was efficient under both low and normal-N conditions. Meanwhile, 20 genotypes were identified as low-N sensitive ones, in which 19 genotypes were inefficient under low-N conditions but efficient under normal-N conditions, one variety was inefficient under both low and normal-N conditions. Conclusion: We preliminarily identified Kashi as a low-N tolerant and N-efficient cotton genotype, and CCRI 64 as a low-N sensitive and N-inefficient cotton genotype. Further studies should be carried out to verify the yield and heritability effect of specific genotypes in the field.