Background: Asparagus racemosus has been attaining its importance due to its high medicinal and pharmaceutical value. This plant resulted in the depletion of its wild population in India. This plant has been placed in the nearly threatened and endangered category of the International Union for Conservation of Nature and Natural Resources (IUCN) in the states of Chhattisgarh, Madhya Pradesh, and West Bengal. The over-demand also poses the problem of adulteration in several ways. Objectives: Selection of elite germplasm for commercial cultivation was done by studying the variation of Shatavarin IV from different geographical regions. The development of simple and reliable high-performance thin layer chromatography (HPTLC) method and chromatographic profile was done for quality control. Materials and Methods: A total of 103 Asparagus racemosus root samples were collected from different agro-climatic zones of Odisha. The methanol root extracts were analyzed qualitatively and quantitatively using HPTLC. The pre-coated silica gel 60 F254 plates were used for the development of chromatograms with ethyl acetate‒methanol-water (7.5:1.5:1, v/v/v) as a mobile phase. Results: The Shatavarin IV was detected at Rf of 0.40 ± 0.05 and showed maximum absorption at 425 nm. The method was validated in terms of linearity, limit of detection (LOD), quantification (LOQ), precision, stability, and recovery test. The amount of Shatavarin IV varied between 0.01-0.40% among different regions. Conclusion: The elite germplasms were identified at Ramagiri Hill sides of Gajapati district, Odisha, having Shatavarin IV content <0.39%. The HPTLC method used here was found to be reliable, precise, and can be used for the quality control assessment of Asparagus racemosus.