The lipid transfer protein (LTP), Pru p 3, has been identified as the major allergen present in peach, and its sequence obtained by direct amino acid sequencing has been previously reported. However, several sequences, obtained from c-DNA and available in databases, show differences among them and from the originally proposed structure. In this paper, we report the fast and unambiguous determination of the structure of Pru p 3 protein, extracted from three different varieties of peach, by electrospray ionization mass spectrometry (ESI-MS), both coupled to single stage (quadrupole) or advanced (FT-HRMS) analyzers. The structure was identical to one of the cDNA-derived sequences and different in two positions from the previously reported structure obtained by amino acid sequencing. Moreover, the exclusive localization of the protein in the outer part of the fruits was assessed by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry Imaging (MALDI MSI). The results reported here demonstrate the full potential of mass spectrometry for rapidly obtaining high quality structural data of relevant food proteins. Copyright © 2009 John Wiley & Sons, Ltd.