20-Hydroxyecdysone (20E) is an insect molting hormone that is also widely spread in various plants. Many chromatographic methods can be used to identify and/or determine 20E content in samples of biological origin and various spectroscopic methods serve to identify its structural elements. We have utilized X-ray crystallography to reveal the stereostructures of 20E. Our data demonstrates that 20E exists in two different crystalline forms that are both orthorhombic modifications. One form is homo-molecular, with a limited freedom of internal rotation of the side chain around the C23-C24 bond and the other, which is a clathrate formed with methanol and water, which minimize the conformational freedom of the side chain.