In this study ten sweet cherry cultivars from Italy were characterized through proteomic analysis of the corresponding fruits hand-harvested at the commercial ripening stage (89 BBCH scale). A TMT10plex-based MS approach was employed to highlight molecular differences between fruit ecotypes in terms of key components influencing fruit quality and allergenic potential. Multivariate analysis of total quantitative data outlined cultivar differences and phenotypic relationships between investigated cultivars. These data could successfully integrate genetic findings obtained with DNA fingerprinting techniques for the elucidation of molecular differences and relationships between fruit ecotypes.