Abstract: Abstract: Leaf photosynthetic characteristics of maize seedlings can greatly contribute to the accuracy of phenotype detection. This study aims to determine the vertical distribution of leaf photosynthesis in three maize varieties at the seedling stage (five-leaf stage). The chlorophyll fluorescence imaging was also utilized to clarify the combined influence of different environmental nitrogen and water content. The results were as follows. 1) The leaf 1 and leaf 2 were not affected by the species, nitrogen, and water treatment, in terms of the chlorophyll fluorescence quenching, indicating the more significantly different fluorescence intensities. The unweighted pair-group method with arithmetic means (UPGMA) cluster analysis was conducted to produce the four levels of differentiation with the leaf position as the dominant factor. The clustering of ZD 958 was less heterogeneous than XY 335 and JD 20 under different environmental factors. There were no outstanding clustering characteristics for the nitrogen and water content. 2) In the vertical distribution of key chlorophyll fluorescence parameters, the parameter Fv/Fm indicated that there was a significant increase in the maximum photosynthetic light energy conversion efficiency of senescent, normal, and neonatal leaves in sequence. There was no variation in the feature within the leaves. The parameter △F/Fm' indicated that the high morphological positions were greatly contributed to the actual light energy conversion efficiency of the two leaves. The parameter qL_Lss indicated that the photoreactive electron transfer activity increased in the three steps with the increase of vertical height. The parameter NPQ_Lss indicated that the less variable non-photochemical energy dissipation was found in the normal leaves, but the significant increase in the senescent leaves. 3) A high fitting characteristics with the quadratic function were found in the vertical distribution of the maximal PSⅡ efficiency (Fv/Fm) at the different ambient nitrogen levels. The heterogeneity of the vertical distribution increased, as the ambient nitrogen level decreased. A high fitting characteristics with the primary function were also found in the vertical distribution of the steady-state light-adapted quenching (qL_Lss) at the different varieties. The heterogeneity of the vertical distribution was ranked in the order of the ZD 958 > XY 335 > JD 20. Among them, the ZD 958 was more resistant to the stress. A comparison was made on the Support Vector Machines (SVM), Random Forest (RF), and Partial Least Squares Discriminant Analysis (PLS-DA). The maximal PSⅡ efficiency (Fv/Fm) of ZD 958, the samples under the trace ambient nitrogen content, and the steady-state non-photochemical quenching (NPQ_Lss) of samples under the drought environment were detected with 0.82, 0.94, and 0.88 accuracy, respectively. There was much more significant pattern of vertical distribution compared to other treatments and fluorescence parameters. 4) The plant leaves were numbered from the bottom to the top in the "key leaf" analysis of leaf photosynthetic heterogeneity. The "key leaf" was then selected as the second leaf, second leaf and first leaf for the identification of maize varieties, the environmental nitrogen, and water content assessment in the discriminant analysis of varieties, water, and nitrogen content treatment, respectively.