Centella asiatica (L.) Urban (CA) has high demand in traditional medicine, skin care, pharmaceutical industries and as a leafy vegetable. India and Madagascar are its key producers. Due to its ever growing demand, CA is collected at an uncontrolled rate from its natural habitats, and this results in destruction of its wild genotypes. Here we report the screening of 106 CA accessions collected from their natural habitats in a wide geographical area in south India for their ASI and MAD contents by HPTLC-densitometry. The edaphic-ecologic parameters of the collection locations of CA accessions were recorded and correlated with their saponin (ASI, MAD) contents. Multilocation trials of CA elite lines at four different agro-climatic locations in Kerala were conducted, and their saponin-biomass yields were determined. Crucially, of these 106 CA accessions, only 6 showed saponin contents above the elite benchmark of industries (ASI+MAD ≥ 4.0%), viz. , 45/Ca-55 (6.18 ± 0.26%), 46/Ca-56 (5.88 ± 0.11%), 48/Ca-58 (4.64 ± 0.15%), 49/Ca-59 (6.94 ± 0.23%), 53/Ca-63 (5.59 ± 0.20%), 55/Ca-65 (5.94 ± 0.18%). Multilocation trials of these elite lines revealed highest saponin contents in Plot 1, characterized by sandy soil (pH 6.0), organic carbon 2.71%, nitrogen 74.20 Kg/ha, phosphorous 100.70 Kg/ha and potassium 23.52 Kg/ha. GC-MS and ICP-MS analyses showed absence of pesticides in CA elite lines and heavy metal contents were within the permissible levels. Highest (ASI+MAD) content (7.55%, dry wt.) and biomass yield (7534.73 Kg/ha) at Plot 1 were shown by the elite line, 49/Ca-59. Average (ASI+MAD) content and biomass yield at Plot 1 were 5.38 ± 1.82% (dry wt.) and 3462.39 ± 2032.54 Kg/ha (n = 6). Noticeably, all six CA elite lines with high ASI, MAD and (ASI+MAD) contents were originally collected from high altitude (700–1700 m) locations. This ASI, MAD screening of CA accessions over a wide geographical area and multilocation trials of elite lines reveal its edaphic and ecological preferences. Our study discovered a hotspot of CA elite clones in south India. This and our previous two reports on a total of 216 accessions provide the range of ASI, MAD and (ASI+MAD) in CA accessions in south India. Further, these studies urge the restriction of random (uncontrolled) collections of CA from its natural habitats, and for the development of cultivation practices of the elite lines for commercial purposes. Overall, this study provides collection, cultivation and conservation strategies towards sustainable management of the resources of this high value medicinal herb. • Centella asiatica accessions (106) from south India were screened for ASI, MAD. • Of 106 CA accessions, only 6 satisfied the elite benchmark of ASI+MAD ≥ 4.0%. • In multilocation trials, elite line showed ASI+MAD 7.55%, biomass 7534.73 Kg/ha. • Altitudes of CA collection locations were correlated to ASI, MAD, ASI+MAD contents. • Discovered a hotspot of CA elite clones in a vast geographical area in south India.