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Centellosides viz., asiatic acid, madecassic acid, asiaticoside, madecassoside, are the major bioactive molecules in Centella asiatica. In this study madecassic acid:asiatic acid, madecassoside:asiaticoside (C6-hydroxylation versus non-hydroxylation) and asiaticoside:asiatic acid, madecassoside:madecassic acid (C28-glycoside versus aglycone) ratios in 50 C. asiatica accessions originally collected from their natural habitats in south India and grown under identical ecological conditions for six generations were determined using validated HPTLC-densitometry protocols. Asiatic acid, madecassic acid, asiaticoside and madecassoside contents ranged from 0.00–0.29% (average 0.03 ± 0.06%; 28 accessions recorded asiatic acid content as zero), 0.02–0.72% (0.12 ± 0.13%), 0.04–2.41% (0.44 ± 0.52%) and 0.15–5.27% (1.59 ± 1.26%), respectively. Distinctly, C6-hydroxylated (madecassic acid:asiatic acid 4.00, madecassoside:asiaticoside 3.61) and C28-glycosylated (asiaticoside:asiatic acid 14.67, madecassoside: madecassic acid 13.25) centellosides dominated over the respective non-derivatized entities. Our results infer that both C6-hydroxylation by CYP450-dependent monooxygenases and C28-glycosylation by UDP-Glc glucosyltransferases are dominant bioconversion steps in C. asiatica. Besides, this study discovered six elite lines of C. asiatica, with their (asiaticoside + madecassoside) contents above the industrial benchmark (≥ 4%) from south India. Two elite clones with asiaticoside contents ≥ 2% were also identified. Standardization of the agrotechniques of these elite lines could lead to their industrial applications. Further, this study emphasizes the need for standardizing all four centellosides as biomarkers in C. asiatica raw drugs, pharmaceutical and cosmetic products.