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• Eighteen spectral indices are compared for their sensitivity to CNC and biomass. • Of these indices, PRI best correlates with CNC but it also correlates with biomass. • Regression relationships between CNC and all indices shift with growth stages. • CCCI exhibits stronger correlation to biomass than CNC. • A wide range of growth conditions are needed to develop a robust indicator for CNC. In precision farming, accurate estimation of canopy nitrogen concentration (CNC) is valuable for effective crop growth monitoring and nitrogen (N) fertiliser management. To date, many canopy multispectral indices have been proposed as indicators for CNC; however, many of these indices have also shown sensitivity to biomass and their performance drops at high biomass levels. Dependence on growth stage, season, or other environmental conditions limits their efficacy as generalized CNC indices. The objectives of this study were to assess the robustness of popular CNC indices across a wide range of biomass levels and fertiliser application levels; and for two contrasting seasons – winter and summer. To achieve this, we analysed the efficacy of seven canopy nitrogen indices, including canopy chlorophyll content index (CCCI), together with eleven other commonly used spectral indices. We used canopy level solar-induced hyperspectral reflectance data acquired using a hand-held optical spectroradiometer across four growth stages in winter (May-June 2018) and four in summer (January-February 2019) from an experimental field of irrigated perennial ryegrass with variable N application in Victoria, Australia. The field contained 40 plots, each with one of eight different N treatments. Almost all the indices exhibited similar correlation to CNC (%) when applied to individual stages (days) in both winter and summer; however, relationships between CNC and individual indices varied significantly between stages. We obtained similar results for canopy biomass. When the data across the entire range of growth stages and seasons were combined, the correlations between most canopy nitrogen indices and CNC became weak (R2 < 0.25, 0.9% ≤ RMSE ≤ 1.0%). PRI exhibited the highest correlation with CNC (R2 = 0.58, RMSE = 0.7%) for the combined data set. Even so, PRI's association with CNC and canopy biomass changed with the season. Most indices responded to both CNC and biomass simultaneously, and this confounds the estimation of CNC due to strong but growth stage-specific relationships between CNC and canopy biomass. This study shows that it is important to consider a wide range of conditions when evaluating multispectral CNC indices. [ABSTRACT FROM AUTHOR]