Development of the new crop cultivars with high yield under low nitrogen (N) input is a fundamental approach to enhance agricultural sustainability, which is dependent on the exploitation of the elite germplasm.  In the present study, four barley genotypes (two Tibetan wild and two cultivated), differing in N use efficiency (NUE), were characterized for their physiological and biochemical responses to different N levels.  Higher N levels significantly increased the contents of other essential nutrients (P, K, Ca, Fe, Cu and Mn), and the increase was more obvious for the N-efficient genotypes (ZD9 and XZ149).  The observation of ultrastructure showed that chloroplast structure was severely damaged under low nitrogen, and the two high N efficient genotypes were relatively less affected.  The activities of the five N metabolism related enzymes, i.e.,  nitrate reductase (NR), glutamine synthetase (GS), nitrite reductase (NiR), glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH) all showed the substantial increase with the increased N level in the culture medium.  However the increased extent differed among the four genotypes, with the two N efficient genotypes showing more increase in comparison with the other two genotypes with relative N inefficiency (HXRL and XZ56).  The current findings showed that a huge difference exists in low N tolerance among barley genotypes, and improvement of some physiological traits (such as enzymes) could be helpful for increasing N utilization efficiency.