Centella asiatica is an important industrial crop in Madagascar that is collected in the wild for its triterpenic glycoside compounds. A multidisciplinary approach was applied to study diversity of 6 populations of C. asiatica. Cytogenetical approach and fluorescent in situ hybridization (FISH) analysis coupled with cytological evaluation of stomata density revealed diploid (2n = 2x = 18) and tetraploid (2n = 4x = 36) populations. Genetic diversity was evaluated through SSR analysis and indicates three separated groups, clearly distinct from Indian and South African genotypes. Chloroplast diversity analysis reveals two chlorotypes corresponding to diploid and tetraploid populations respectively, indicating an ancient origin of polyploidy. Triterpenic glycoside content of some clones was evaluated confirming that active compound content is highly variable. Implications of our results for further genetic studies and set-up breeding program are discussed.