The nitrogen isotope composition (δ15N) of plants has potential to provide time‐integrated information on nitrogen uptake, assimilation and allocation. Here, we take advantage of existing T‐DNAandγ‐ray mutant lines ofArabidopsis thalianato modify whole‐plant and organ‐level nitrogen isotope composition. Nitrate reductase 2 (nia2), nitrate reductase 1 (nia1) and nitrate transporter (nrt2) mutant lines and the Col‐0 wild type were grown hydroponically under steady‐stateNO3–conditions at either 100 or 1000 μMNO3–for 35 days. There were no significant effects on whole‐plant discrimination and growth in the assimilatory mutants (nia2andnia1). Pronounced root vs leaf differences inδ15N, however, indicated thatnia2had an increased proportion of nitrogen assimilation ofNO3–in leaves whilenia1had an increased proportion of assimilation in roots. These observations are consistent with reported ratios ofnia1andnia2gene expression levels in leaves and roots. Greater whole‐plant discrimination innrt2indicated an increase in efflux of unassimilatedNO3–back to the rooting medium. This phenotype was associated with an overall reduction inNO3–uptake, assimilation and decreased partitioning ofNO3–assimilation to the leaves, presumably because of decreased symplastic intercellular movement ofNO3–in the root. Although the results were more varied than expected, they are interpretable within the context of expected mechanisms of whole‐plant and organ‐level nitrogen isotope discrimination that indicate variation in nitrogen fluxes, assimilation and allocation between lines.