This study reveals the diversity of salvianolic acid (SA) biosynthesis in Salvia miltiorrhiza, a medicinal plant rich in SA. SA, including rosmarinic acid (RA), danshensu (DSS), and their derivatives, are widely found in Lamiaceae and Boraginaceae plants. Previous studies have identified 4-coumaroyl-CoA and 4-hydroxyphenyllactic acid (4-HPL) as common substrates for SA biosynthesis, with the involvement of rosmarinic acid synthase (RAS) and cytochrome P450 98A (CYP98A) enzymes. However, this study shows that various acyl donors and acceptors, including DSS and its ester-forming products, are present in SA-rich plants, indicating that previous understanding of SA biosynthesis is incomplete. Using S. miltiorrhiza, a representative SA-rich plant, the study elucidates the diversity of SA biosynthesis. SmRAS catalyzes various acyl donors and acceptors to form precursors of RA, while SmCYP98A family members, SmCYP98A14 and SmCYP98A75, are responsible for different positions' meta-hydroxylation of these precursors. SmCYP98A75 preferentially catalyzes C-3' hydroxylation, and SmCYP98A14 preferentially catalyzes C-3 hydroxylation in RA generation. Additionally, SmCYP98A75 has been verified as the first enzyme that participates in DSS formation. Knockout of SmCYP98A enzymes resulted in decreased SA accumulation, while overexpression increased SA content, indicating the importance of these enzymes in SA biosynthesis. The study provides new insights into the diversity of SA biosynthesis in SA-abundant species and the versatility of CYP98A enzymes in catalytic preference for meta-hydroxylation reactions. CYP98A enzymes are ideal targets for metabolic engineering to increase SA content. The study also highlights the importance of understanding the molecular mechanisms of SA biosynthesis and the function of CYP98A enzymes in this process. The findings contribute to the understanding of SA biosynthesis and have potential applications in the pharmaceutical and food industries.This study reveals the diversity of salvianolic acid (SA) biosynthesis in Salvia miltiorrhiza, a medicinal plant rich in SA. SA, including rosmarinic acid (RA), danshensu (DSS), and their derivatives, are widely found in Lamiaceae and Boraginaceae plants. Previous studies have identified 4-coumaroyl-CoA and 4-hydroxyphenyllactic acid (4-HPL) as common substrates for SA biosynthesis, with the involvement of rosmarinic acid synthase (RAS) and cytochrome P450 98A (CYP98A) enzymes. However, this study shows that various acyl donors and acceptors, including DSS and its ester-forming products, are present in SA-rich plants, indicating that previous understanding of SA biosynthesis is incomplete. Using S. miltiorrhiza, a representative SA-rich plant, the study elucidates the diversity of SA biosynthesis. SmRAS catalyzes various acyl donors and acceptors to form precursors of RA, while SmCYP98A family members, SmCYP98A14 and SmCYP98A75, are responsible for different positions' meta-hydroxylation of these precursors. SmCYP98A75 preferentially catalyzes C-3' hydroxylation, and SmCYP98A14 preferentially catalyzes C-3 hydroxylation in RA generation. Additionally, SmCYP98A75 has been verified as the first enzyme that participates in DSS formation. Knockout of SmCYP98A enzymes resulted in decreased SA accumulation, while overexpression increased SA content, indicating the importance of these enzymes in SA biosynthesis. The study provides new insights into the diversity of SA biosynthesis in SA-abundant species and the versatility of CYP98A enzymes in catalytic preference for meta-hydroxylation reactions. CYP98A enzymes are ideal targets for metabolic engineering to increase SA content. The study also highlights the importance of understanding the molecular mechanisms of SA biosynthesis and the function of CYP98A enzymes in this process. The findings contribute to the understanding of SA biosynthesis and have potential applications in the pharmaceutical and food industries.