Genetic diversity can remarkably impact the quality of derived products. This study focused on Adenophora triphylla, an herbaceous perennial used in East Asian traditional medicine. It is known for its diverse therapeutic effects that are attributed to phytochemicals. However, despite its therapeutic potential, a comprehensive exploration of the influence of genetic diversity on the pharmaceutical properties of various A. triphylla genotypes remains unknown. To address this knowledge gap, we employed DNA barcoding regions (ITS2, matK, and psbA-trnH) to elucidate the genetic diversity among the collected genotypes. The combined application of these barcoding regions is a robust method to identify A. triphylla genotypes. In addition, leaf extracts consistently exhibited higher antioxidant potential than root extracts. Nitric oxide production was strongly inhibited by the leaf extracts of the BG genotype (IC₅₀ = 124.52 ± 12.97 µg/mL), and the leaf extracts (200 µg/mL) of the BT genotype contained significant antimelanogenic properties (IC₅₀ = 181.56 ± 16.17 µg/mL). Various genotypes displayed diverse levels of phytochemical content, and the total saponin content exhibited a strong correlation with various biological activities of A. triphylla, suggesting a genetic influence on their pharmaceutical attributes. These findings highlight the significance of genotype selection for optimizing health benefits and promoting the potential utilization of specific genotypes in various industries, such as cosmetics and pharmaceuticals.