20-hydroxyecdysone (20E) is a biologically active compound, belonging to the ecdysteroid group, which is commonly found in plants. The ecdysteroids extracted from plants have many important pharmacological properties; products containing ecdysteroids have been widely used in pharmaceutical medicine and agriculture. However, researches and applications of ecdysteroids in Vietnam are very limited. In this study, the 20E contents of 167 medicinal plant species have been collected and analysed in Tam Dao National Park and the buffer zone. The results have revealed 45 species containing 20E out of 167 species, accounting for 26.96%. The contents of 20E in the species ranged from 0.002 to 0.588% of dry matter; the highest content was found in Croton tonkinensis, that is 0.588%. 20E concentrated in leaves and buds of most species. Among of the 45 species, 28 species have not been published on 20E worldwide, 7 species belong to the genera which contained the published species, and only 10 species were published. These results exhibit the diversity and value of medicinal plants containing 20E in Tam Dao National Park and the buffer zone, contributing to the conservation and sustainable development of plant resources.