Nitrogen (N) deprivation or excess can lead to dramatic phenotype change, disrupt important biological processes, and ultimately limit plant productivity. To explore genes in Tibetan hulless barley responsive to varied N stress, we utilized a comparative transcriptomics method to investigate gene expression patterns under three nitrate treatments. The transcriptome of the control (optimal-nitrate, ON) sample was compared with that of free-nitrate (FN), low-nitrate (LN) and high-nitrate (HN) treatment samples, identifying 2,428, 1,274, and 1,861 genes, respectively, that exhibited significant differences in transcript abundance. Among these, 9 genes encoding ribulose bisphosphate carboxylases exhibited up-regulated expression under varied N stress. We further compared FN versus ON and LN versus ON to investigate the impact of stress degree on gene expression. With the aggravation of stress, more genes were differentially expressed and thus possibly involved in the response to nitrogen deficiency. Cluster and functional enrichment analysis indicated that the differentially expressed genes (DEGs) in FN were highly enriched in response to stress, defense response, and gene expression regulation. Comprehensive comparison analysis further suggested that Tibetan hulless barley could respond to varied N stress by regulating multiple common biological processes and pathways such as nitrogen metabolism, carbon metabolism, and photosynthesis. A large number of specific DEGs involved in diverse biological processes were also detected, implying differences in the potential regulatory patterns of low- and high-N stress response. Notably, we also identified some NIN-like proteins and other transcription factors significantly modulated by these stresses, suggesting the involvement of these transcription factors in N stress response. To our knowledge, this study is the first investigation of the Tibetan hulless barley transcriptome under N stress. The identified N-stress-related genes may provide resources for genetic improvement and promote N use efficiency.