Drought stress significantly threatens tea production and quality worldwide. To elucidate the genetic basis of drought tolerance in tea plant, we evaluated 11 physiological traits across 115 diverse tea accessions under drought conditions. A comprehensive drought resistance index (D-value) was constructed through principal component analysis and fuzzy membership function. Genome-wide association studies identified 67 significant SNPs and pinpointed four candidate genes associated with drought-responsive traits. Integrated transcriptome and qRT-PCR analyses revealed that three genes, including CsAGD6, were significantly upregulated under drought stress. Functional assays confirmed that CsAGD6, encoding a nucleus-localized ARF-GAP protein, positively regulates drought tolerance by modulating photosynthetic efficiency and membrane stability. Haplotype analysis identified favorable alleles Hap-P1 and Hap-C1 in the promoter and coding regions of CsAGD6, respectively. Moreover, an SNP-kompetitive allele-specific PCR marker targeting chr10:206216541 (C/T) was developed and validated in 104 accessions, demonstrating high efficacy for early selection of drought-tolerant genotypes. This study provides novel insights into the molecular mechanisms of drought tolerance in tea plant and offers valuable genetic resources and tools for marker-assisted breeding.

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