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Hydrolyzed tannin 1,2,6-tri-O-galloyl-β-D-glucopyranose (1,2,6-TGGP) possesses significant medicinal properties. However, little is known about its underlying molecular mechanisms. In this study, the levels of 1,2,6-TGGP in tea materials from different cultivars and leaf positions were compared. Additionally, one leaf and one bud sample from six tea cultivars with significant variations in 1,2,6-TGGP levels were analyzed using transcriptome high-throughput sequencing to identify the genes that are responsible for 1,2,6-TGGP accumulation. The sequencing results were mapped to the reference tea genome, revealing a total of 2735 differentially expressed genes (DEGs). This set included four UDP glycosyltransferase (UGTs) and six serine carboxypeptidases-like (SCPLs) genes. Among them, the upregulated SCPLs (<i>CSS0032817</i>) may directly participate in the acylation reaction of 1,2,6-TGGP. In addition, several classes of DEGs, including cytochrome P450, were significantly associated with the 1,2,6-TGGP content, which is potentially involved in their regulation. Overall, these results provide new insights into the molecular mechanism of 1,2,6-TGGP accumulation.