In order to explore the path of nitrogen input to grains after anthesis, GS inhibitor (Glufosinate, GLF) and 15N tracer technique at different growth stages under different nitrogen levels were applied to investigate the characteristics of inorganic nitrogen assimilation in organs of two wheat cultivars with varied grain protein contents. The results showed that NDFF (percentage of organ N from soil nitrogen) in spike of Yumai47 significantly increased, while that of Yumai50 significantly decreased by the treatment of leaf GS inhibitor. NDFF in leaves of Yumai47 increased, and that of Yumai50 increased at low nitrogen rate and decreased at high nitrogen rate by the treatment of spike GLF. The amount of inorganic nitrogen assimilation in Yumai47 was greater than that in Yumai50 after anthesis. The main organs for assimilating inorganic nitrogen were the roots and stems for Yumai47, and leaves for Yumai50. The ratios of the inorganic nitrogen assimilation by the roots and stems, the leaves, and the spikes were about 4∶1∶2 for Yumai47, and about 1∶5∶1 for Yumai50. With increased nitrogen application, the amount of inorganic nitrogen assimilation in Yumai47 increased gradually after anthesis, while that of Yumai50 decreased. Furthermore, the output of nitrogen assimilated by the leaves of Yumai47 was significantly less than that by the grain, yet the opposite pattern was seen with Yumai50. The results suggested that there could be different paths for nitrogen transport from the roots to the grains after anthesis: that of high grain protein genotype Yumai47 did not have to go through the leaves, but that of low grain protein genotype Yumai50 had to go through the leaves.