Ajuga bracteosa is an endangered medicinal herb which contains several natural products of therapeutic importance like 20-hydroxyecdysone (20-HE). As geography and habitat play a crucial role in the metabolism and morphology of a plant, the present study was aimed at evaluating the impact of phytogeography, season and tissue type on morphology and 20-HE content of A. bracteosa. The results revealed large morphological variations in various ecotypes of A. bracteosa. However, plants from the same altitude, regardless of their phytogeography, represented similar morphology. Effect of habitat on 20-HE content remained non-significant except for Karot (1608μg/g) and Kahuta (728μg/g). Effect of tissue types was significant (p value <0.016) for 20-HE content and followed ascending order: root<stem<leaf<flower, representing the tender aerial tissues' hormonal supremacy. Seasons showed a significant impact (p value <0.001) on 20-HE content with the pattern: winter (1902μg/g>>spring (1071μg/g>>summer (617μg/g). The aerial tissue types contained more 20-HE content in all seasons; especially during winter its amount radically rose in flowers (μ=2814μg/g). The aerial portion of Karot ecotype harvested in winter offers a valuable source of 20-HE. To confirm the effect of low temperature on 20-HE content, profiling of A. bracteosa raised in vitro at different temperature regime was carried out. On the basis of these results we hypothesize that chilling cold hampers vegetative growth and triggers stress induced 20-HE accumulation as a defense response.