Abstract: In order to investigate the metabolic characteristics of nitrogen nutrition in cotton, and to give some references for breeding cotton varieties with higher nitrogen efficiency, 30 cotton varieties were cultured by floating seedling method and fertilized by two N levels (0 mmol/L and 20 mmol/L), a comparative study of their seedling nitrogen efficiency in this study, and yield and fiber quality of all varieties were also determined. The results show that nitrogen stress can lead to a significant decrease in dry weight, shoot ratio, chlorophyll content, nitrogen content, nitrogen accumulation and chlorophyll saturated ETR values. The main component analysis based on 20 indicators of seedling indicate that the cumulative variance contribution ratio of the first three principal components reached 80% above of all indicators, and the six indicators including relative dry weight, total nitrogen accumulation amount, nitrogen content of aboveground, aboveground relative nitrogen content, nitrogen content of underground part and underground part relative nitrogen content can represent most information of all these traits. According to cluster analysis of these 30 cotton varieties based on these six indicators at seedling stage, and cluster analysis based on yield and fiber quality traits, a comprehensive analysis of these three different cluster analysis results shows that nitrogen efficiency at seedling stage can effect yield and fiber quality to a certain extent. The varieties with higher nitrogen efficiency at seedling stage, such as ‘Emian 14’, ‘Simian 4’, ‘Xiangmian 10’, ‘Hengmian 4’ and ‘Chuanmian 56’, have a higher yield traits and fiber quality, while the lower varieties including ‘Lumian 12’, ‘Liaomian 15’ and ‘Xinluzao 20’, have a lower yield traits and fiber quality.