The phenolic profile of strawberry fruits (Fragaria x ananassa Duch., Rosaceae) was investigated by high-performance liquid chromatography with photodiode array detection. A peak displaying retention time and UV spectral data identical to those of phloridzin (phloretin 2'-O-beta-d-glucoside), a dihydrochalcone glucoside so far considered characteristic of apples, was monitored. For further characterization, crude extracts of strawberries were purified on polyamide, and the target compound was isolated by preparative and analytical HPLC. Structure elucidation was performed on the basis of APCI- and ESI-MS in the negative ion mode as well as by 1D and 2D NMR spectroscopy using authentic phloridzin for comparison. The d-configuration of the sugar moiety was established by HPLC analysis of the corresponding acyclic 1-deoxy-1-(N-acetyl-alpha-methylbenzylamino)alditol acetate. Apart from its chemotaxonomic relevance, this first report on the occurrence of phloridzin in strawberries is of particular interest for the authenticity control of strawberry products such as juices, jams, and fruit preparations since phloridzin has so far been used for the detection of fraudulent admixtures.