Spinach (Spinacia oleracea L.) is a short-cycle leafy crop that has a high demand for nitrogen in order to rapidly come to a harvestable product. To increase sustainability of spinach growers need cultivars with a satisfactory yield under reduced N input conditions. Nitrogen use efficiency (NUE), defined as the ability to produce high biomass per unit N applied, is low in spinach. The present study aims to evaluate spinach genotypes for selectable traits under varying N supply and provide tools and knowledge to facilitate the development of varieties with good yield, quality and stability under low N input. The results of the study comprised: i) genetic diversity for NUE related traits; ii) the effect of N application strategy in a single bulk or a steady-state application; iii) development of tools for molecular genetic evaluation of NUE in spinach; and iv) an in depth genotype by environment interaction analysis using several environmental factors. The studies presented in this thesis resulted in knowledge and tools that can be implemented in efficient breeding strategies for the complex trait nitrogen use efficiency.