Plants synthesize various types of triterpenoids as secondary metabolites to achieve greater fitness with respect to their surrounding environment. In last few decades, interest in biochemical synthesis and regulation of plant triterpenoids has been increased due to their scientific and economic significance. However, until now evolution of such triterpenoid synthesis over geological time is not clearly understood. In order to better understand the biosynthesis of triterpenoid in deep time, the present study reports hopenol-B (hop-22[29]-en-3β-ol), a plant secondary metabolite from fossil dammar resin of Eocene time, derived from Dipterocarpaceae family. The studied fossil resins were obtained from two lignite-bearing sequences of early and middle Eocene age from the Cambay and Kutch Basin, western India, respectively. This bioactive metabolite is recorded from several modern angiosperm trees. Hopenol-B is synthesized by the enzyme (oxidosqalene cyclase containing hopanoid skeleton)-mediated cyclization of 2,3-oxidosqualene by which other angiosperm-derived triterpenoids are also formed. In contrast, hopanoid class triterpenoids of bacterial and non-vascular plants origin are formed by the cyclization of squalene, catalyzed by squalene-hopene cyclases (SHCs). The present study provides the earliest signature of hopenol-B found so far and its occurrence in the analyzed samples suggests that the biosynthetic pathway of this compound in angiosperms had already been evolved during the early Eocene.