Ashwagandha [Withania somnifera (L.) Dunal] is a highly valued medicinal plant in Ayurveda, used either singly or in combination with other herbs. Plants growing under different agro-climatic condition often show qualitative and quantitative variations in their phytoconstituents. In the GC-MS analysis, 24 bioactive phytochemical compounds were identified in roots and leaves of Withania somnifera. Present investigation deals with comparison of Ashwagandha plants (WsL, WsK, WsN and WsM) obtained from various regions (Lucknow, Karnataka, Nimuch and Mumbai) of India, for their withanolide-A contents using HPTLC. Methanolic root extracts of WsN showed maximum withanolide-A content (1.3%), WsK being close behind at 1.2% followed by WsM (0.7%); WsL showed minimum withanolide-A content at 0.4% only.