Background: Costus speciosus known as "insulin plant" for its anti-diabetic potential. It has commercial significance due to the presence of industrially viable metabolite diosgenin. Objective: The identification of elite chemotypes of C. speciosus (Rhizome) through high-performance thin-layer chromatography (HPTLC) from the Eastern Ghats (India). Materials and Methods:A validated HPTLC method for the quantification of diosgenin was developed in accordance with the International Conference on Harmonization Guidelines. Results: In total, 11 populations of species were collected from their natural habitat with all the Global Positioning System (GPS) coordinates. The method was developed on HPTLC pre-coated silica gel 60 F254plates under a binary solvent system of n-hexane and ethyl acetate (7:2 v/v). The linearity was established at concentration range of 0.1–0.9 μg/spot having regression equation, 0.010× + 0.002. The limit of detection and limit of quantification were 0.907 and 2.751 with a regression coefficient of 0.999. The diosgenin content varies significantly (P < 0.05) from 0.002% to 0.076% and NBCS-06 from Patiya, Bhubaneswar, was identified as elite chemotype. Conclusion: The validation data confirm that developed HPTLC method was precise, accurate, robust, reproducible, and reliable in nature. The study resulted in the identification of elite chemotype of C. speciosus through validated HPTLC method from the Eastern Ghats of India. It will promote site-specific commercial cultivation of high metabolite yielding germplasm for good quality raw material to meet the industrial demand and in turn income generation of local inhabitants. The developed method will also aid in the regulation of quality standard and batch consistency of diosgenin-containing formulations in industry.