The title compound {systematic name: 4-[(3R)-8,8-dimethyl-3,4-dihydro-2H-pyrano[2,3-f]chromen-3-yl]benzene-1,3-diol, commonly named glabridin}, C₂₀H₂₀O₄, is a species-specific biomarker from the rootsGlycyrrhiza glabraL. (European licorice, Fabaceae). In the present study, this prenylated isoflavan has been purified from an enriched CHCl₃fraction of the extract of the root, using three steps of medium-pressure liquid chromatography (MPLC) by employing HW-40F, Sephadex LH-20 and LiChroCN as adsorbents. Pure glabridin was crystallized from an MeOH–H₂O mixture (95:5 v/v) to yield colorless crystals containing one molecule per asymmetric unit (Z′ = 1) in the space groupP2₁2₁2₁. Although the crystal structure has been reported before, the determination of the absolute configuration remained uncertain. Stereochemical analysis, including circular dichroism, NMR data and an X-ray diffraction data set with Bijvoet differences, confirms that glabridin, purified from its natural source, is found only in a C3Rconfiguration. These results can therefore be used as a reference for the assignment of the configuration and enantiopurity of any isolated or synthetic glabridin sample.