The secondary metabolites extracted from both in vivo and in vitro propagated Crotalaria species were comparatively estimated. The in vitro propagated Crotalaria species were obtained from the explants of three medicinally important Crotalaria species (C.prostrata, C. retusa and C. medicagenea) on Murashige and Skoog (MS) medium fortified with variant concentrations of growth regulators (6-Benzylaminopurine (BAP), α – Naphtahlene acetic acid (NAA), 2, 4-Dichlorophenoxy acetic acid (2,4-D) and Kinetin). An optimal response of 12.6 shoots per explant (6.2 cm length) was obtained from Crotalaria retusa on Murashige and Skoog medium fortified with 13.31 µM 6-Benzylaminopurine and 2.15 µM α – Naphthalene acetic acid. The shoots raised were rooted optimally on Murashige and Skoog medium containing Indolebutyric acid (7.38 µM) with 4.4 roots per shoot. Rooted plantlets thus developed were transferred to greenhouse after hardening with a mix of soil and compost (1:1). Nearly 90% of in vitro raised plants of Crotalaria species were acclimatized. The growth and morphology of in vitro regenerated plants resemble wild species. Therefore in vitro propagated plants of all three species were subjected to comparative estimation of secondary metabolites with both callus cultures and wild species of Crotalaria. Obtained optimal alkaloid content with 29.0% per gram of leaf dry weight was for in vitro propagated Crotalaria retusa amongst the three. Hence from the present investigation it was proved that the quantity of secondary metabolites of an in vitro propagated Crotalaria species is higher than field grown for pharmaceutical preparations.