ABSTRACT: Improving nitrogen use efficiency (NUE) represents one of the main goals to reduce N input in maximizing crop yield for sustainable agriculture. A NUE key strategy is the exploitation of genetic variation in available germplasm together with the understanding of molecular mechanisms governing this complex trait. Thus, NUE, its components, nitrogen uptake efficiency (NUpE) and nitrogen utilization efficiency (NUtE), and NUE-related traits heritability were evaluated in ancient (Cappelli, Capeiti, Russello, and Mazzancoio) and modern (Messapia, Tiziana, Svevo, and Normanno) wheat genotypes for tackling nitrogen (N) and/or water limitation in both growth chamber and field experiments. Our results exhibited a reduction of NUE, NUpE, and NUtE under water and combined (nitrogen + water) stress in all the genotypes, as expected. The contribution of genetic variability on phenotypic variation was significant for NUtE, harvest index, post-anthesis nitrogen uptake (PANU), and biomass production traits. Moreover, the stress tolerance indexes, calculated and bi-plotted for N and water stresses, exhibited two distinct clusters for many traits as then confirmed by principal component analysis. Although modern varieties showed higher crop yield and NUE under conventional N and water regimes, ancient varieties exhibited best performances to cope with both stresses, mainly under water limitation. Finally, the usage index, which takes into account total biomass increase, underlined that old genotypes were less affected by both stresses during crop cycle. In particular, these genotypes showed the best performances for NUE and its components under both stresses at stem elongation and milk ripening as shown also by PANU. In addition, at these stages, nitrate and ammonium transporter gene expressions in the root were performed, showing the highest activity in ancient varieties. In conclusion, the identification of NUE traits during a specific crop cycle stage, under both N and water limitation, will help in the breeding of more resilient varieties in Mediterranean sustainable agriculture by reducing N supply.