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Protecting the environment by improving the crop-system nitrogen (N) use efficiency (NUE) while maximising yield and quality is a primary challenge for modern agriculture, and understanding the processes that govern N fluxes in the plant-soil system is essential to improve NUE. An on-farm study was conducted over two fall-winter seasons to evaluate the NUE, agronomical and physiological response of romaine (var. longifolia, cv Manavert) and red oak-leaf (var. crispa, cv Aruba) lettuce (Lactuca sativa L.) to different N-rates (0, 60, 120, 180 kg ha–1 of N). Nitrogen rate influenced all tested parameters, including plant fresh and dry weight, N accumulation, leaf NO3– and dry matter content, NUE indices, N nutrition index (NNI), soil residual N and the estimated N losses at the end of the crop season. Fresh yield, dry weight and N-accumulation response to N rate were influenced by lettuce genotype. Manavert had higher N recovery, NUE, and lower leaf NO3– concentration than Aruba. Analysing the NNI overtime, 120 kg ha–1 of N assured an optimal N status in both Manavert and Aruba, while N deficiency and excess were observed at lower and higher N-rates, respectively. An empirical relationship was observed between NNI and leaf NO3– concentration, suggesting that leaf NO3– concentration may be used to predict NNI and thus the crop N status. The relationship between NNI and leaf NO3– concentration may be used to define optimal leaf NO3– concentration ranges for the rapid and site-specific assessment of the crop N status, and the dynamic adjustment of N-fertilisation, contributing to improve crop NUE, minimise N-losses, and optimise yield and quality of lettuce crops.