Extracts of apple fruits (Malus domestica cv. "Brettacher") were analysed by HPLC with photodiode array detection. An unknown peak was monitored displaying the same retention time as isorhamnetin 3-O-glucoside. Preliminary identification of the isorhamnetin aglycone was performed by comparison of UV spectral data of the unknown compound with a reference substance. Using atmospheric pressure chemical ionisation mass spectrometry in the negative ion mode, the presence of an isorhamnetin glycoside was supported by loss of 162 amu from the pseudomolecular ion (m/z 477). MS2 product ion analysis of the parent ion m/z 477 provided a fragmentation pattern identical to the reference. Collision-induced dissociation of the aglycone (m/z 315) in the MS3 product ion analysis allowed the differentiation of rhamnetin and isorhamnetin, and unambiguous assignment by comparison with standard compounds. A second isorhamnetin glycoside eluting prior to the glucoside was tentatively identified as isorhamnetin 3-O-galactoside. To the best of our knowledge, this is the first report of isorhamnetin glycosides in apple fruit extracts. Results are discussed with respect to chemotaxonomic relevance within the genera Malus and Pyrus, and especially in consideration of the control of the authenticity of apple products.