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The molecular mechanisms underlying the variation in N-use efficiency (NUE) in pepper (Capsicum annuum L.) genotypes are poorly understood. In this work, two genotypes (750-1, low-N tolerant; ZCFB, low-N sensitive) with contrasting low-N tolerance were selected from 100 pepper cultivars on the basis of their relative leaf areas, shoot dry weights, root dry weights, and plant dry weights at the seedling stage. Subsequently, using RNA-Seq, the transcriptome of these two pepper genotypes under N starvation for 28 days was analyzed. We detected 2621/2470 and 3936/4218 different expressed genes (DEGs) in the leaves/roots of 750-1 and ZCFB, respectively. The changes in the expression of basic N metabolism genes were similar between 750-1 and ZCFB. However, different DEGs not directly involved in N metabolism were identified between the 750-1 and ZCFB cultivars. In 750-1, 110 unique DEGs were detected in the leaves, of which 103 were down-regulated, including genes associated with protein metabolism, photosynthesis, secondary metabolism, cell wall metabolism, stress response, and disease resistance. In ZCFB, 142 unique DEGs were detected in the roots, of which 117 were up-regulated, resulting in enhancement of processes such as protein degradation, secondary metabolites synthesis, lipid metabolism, endocytosis, the tricarboxylic acid cycle (TCA), transcriptional regulation, stress response, and disease resistance. Our results not only facilitate an understanding of the different regulatory process in low-N-tolerant and low-N-sensitive pepper cultivars, but also provide abundant candidate genes for improving the low-N tolerance of pepper cultivars. Full article