Withania somnifera, a perennial plant belonging to the family Solanaceae also known in India as Ashwagandha or winter cherry which is used for preparing more than 100 herbal drugs acts as a cure of several important diseases like cancer, arthritis, gynecological disorders, fungal infection, diuresis and tuberculosis.Variation inmorphological nature of thismedicinally important plant has already been reported. In the present study an attempt has been made to study the variation in morphological nature of Withania somnifera based on the biochemical constituents of Withania somnifera roots collected from various ecological locations includes Dharapuram, Nagpur, Calicut, Udaipur, Kallakurichi and Dindigul. Quantification of biochemical constituents revealed the variation among the samples collected from different locations. Moreover the sample from Nagpur (WSN1r) with minimumstarch content (286±0.017 mg/g) considered as best for preparing commercial drugs. Methanolic extracts of all samples upon phytochemical analysis showed the presence of proteins, amino acids, sugars, starch, saponins and alkaloids. TLC analysis performed using methanol extracts of samples with the solvent system chloroform: methanol = 18:2 revealed the spots with various Rf values indicating the existence of chemotypes with the varied withanolide content.