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Nitrogen (N) is an essential factor for crop yield. Here, we characterized 605 genes from 25 gene families that form the complex gene networks of N utilization pathway in Brassica napus. We found unequal gene distribution between the An- and Cn-sub-genomes, and that genes derived from Brassica rapa were more retained. Transcriptome analysis indicated that N utilization pathway gene activity shifted in a spatio-temporal manner in B. napus. A low N (LN) stress RNA-seq of B. napus seedling leaves and roots was generated, which proved that most N utilization related genes were sensitive to LN stress, thereby forming co-expression network modules. Nine candidate genes in N utilization pathway were confirmed to be significantly induced under N deficiency conditions in B. napus roots, indicating their potential roles in LN stress response process. Analyses of 22 representative species confirmed that the N utilization gene networks were widely present in plants ranging from Chlorophyta to angiosperms with a rapid expansion trend. Consistent with B. napus, the genes in this pathway commonly showed a wide and conserved expression profile in response to N stress in other plants. The network, genes, and gene-regulatory modules identified here represent resources that may enhance the N utilization efficiency or the LN tolerance of B. napus.