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Background Breeding programs for nutrient-efficient tea plant varieties could be advanced by the combination of genotyping and phenotyping technologies. This study was aimed to search functional SNPs in key genes related to the nitrogen-assimilation in the collection of tea plant Camellia sinensis (L.) Kuntze. In addition, the objective of this study was to reveal efficient vegetation indices for phenotyping of nitrogen deficiency response in tea collection. Methods The study was conducted on the tea plant collection of Camellia sinensis (L.) Kuntze of Western Caucasus grown without nitrogen fertilizers. Phenotypic data was collected by measuring the spectral reflectance of leaves in the 350–1100 nm range calculated as vegetation indices by the portable hyperspectral spectrometer Ci710s. Single nucleotide polymorphisms were identified in 30 key genes related to nitrogen assimilation and tea quality. For this, pooled amplicon sequencing, SNPs annotation and effect prediction with SnpEFF tool were used. Further, a linear regression model was applied to reveal associations between the functional SNPs and the efficient vegetation indices. Results PCA and regression analysis revealed significant vegetation indices with high R2 values (more than 0.5) and the most reliable indices to select ND-tolerant genotypes were established: ZMI, CNDVI, RENDVI, VREI1, GM2, GM1, PRI, and Ctr2, VREI3, VREI2. The largest SNPs frequency was observed in several genes, namely F3’5’Hb, UFGTa, UFGTb, 4Cl, and AMT1.2. SNPs in NRT2.4, PIP, AlaDC, DFRa, and GS1.2 were inherent in ND-susceptible genotypes. Additionally, SNPs in AlaAT1, MYB4, and WRKY57, were led to alterations in protein structure and were observed in ND-susceptible tea genotypes. Associations were revealed between flavanol reflectance index (FRI) and SNPs in ASNb and PIP, that change the amino acids. In addition, two SNPs in 4Cl were associated with water band index (WBI). Conclusions The results will be useful to identify tolerant and susceptible tea genotypes under nitrogen deficiency. Revealed missense SNPs and associations with vegetation indices improve our understanding of nitrogen effect on tea quality. The findings in our study would provide new insights into the genetic basis of tea quality variation under the N-deficiency and facilitate the identification of elite genes to enhance tea quality.