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Improving nitrogen (N) management of small-scale farming systems in developing countries is crucially important for food security and sustainable development of world agriculture, but it is also very challenging. The N Nutrition Index (NNI) is a reliable indicator for crop N status, and there is an urgent need to develop an effective method to non-destructively estimate crop NNI in different smallholder farmer fields to guide in-season N management. The eBee fixed-wing unmanned aerial vehicle (UAV)-based remote sensing system, a ready-to-deploy aircraft with a Parrot Sequoia+ multispectral camera onboard, has been used for applications in precision agriculture. The objectives of this study were to (i) determine the potential of using fixed-wing UAV-based multispectral remote sensing for non-destructive estimation of winter wheat NNI in different smallholder farmer fields across the study village in the North China Plain (NCP) and (ii) develop a practical strategy for village-scale winter wheat N status diagnosis in small scale farming systems. Four plot experiments were conducted within farmer fields in 2016 and 2017 in a village of Laoling County, Shandong Province in the NCP for evaluation of a published critical N dilution curve and for serving as reference plots. UAV remote sensing images were collected from all the fields across the village in 2017 and 2018. About 150 plant samples were collected from farmer fields and plot experiments each year for ground truthing. Two indirect and two direct approaches were evaluated for estimating NNI using vegetation indices (VIs). To facilitate practical applications, the performance of three commonly used normalized difference VIs were compared with the top performing VIs selected from 59 tested indices. The most practical and stable method was using VIs to calculate N sufficiency index (NSI) and then to estimate NNI non-destructively (R2 = 0.53–0.56). Using NSI thresholds to diagnose N status directly was quite stable, with a 57–59% diagnostic accuracy rate. This strategy is practical and least affected by the choice of VIs across fields, varieties, and years. This study demonstrates that fixed-wing UAV–based remote sensing is a promising technology for in-season diagnosis of winter wheat N status in smallholder farmer fields at village scale. The considerable variability in local soil conditions and crop management practices influenced the overall accuracy of N diagnosis, so more studies are needed to further validate and optimize the reported strategy and consecutively develop practical UAV remote sensing–based in-season N recommendation methods. [ABSTRACT FROM AUTHOR]