•Each berry fruit species has unique protein composition of seeds.•Different varieties can differentiate by the abundance of their polypeptides.•Image analysis allowed detection and quantification of polypeptides in seeds.•Advanced data-analysis with SDS-PAGE could be used for authentication assessment. Product authentication is one of the most important food quality assurances. Considering the importance of consumption of berry fruits with proven health-beneficial properties, high sensory values and rich composition in bioactive substances, the aim of this study was to evaluate a straightforward and simple procedure for the protein fingerprinting of berry seeds. For this purpose, protein profiles of 45 samples of genuine berry fruit cultivars (strawberry, raspberry, blackberry, black currant, blueberry, gooseberry, chokeberry, cape gooseberry, and gojiberry) were analyzed by SDS-PAGE electrophoresis in combination with advanced chemometric tools. The most important parameters for discrimination among berry seeds were polypeptides at 12.8; 15.1; 25.0; 26.4; 30.0; 41.8; 44.4; 46.0; 48.5; 52.3 and 56.4 kDa. Biomarkers obtained from the protein profile of berry seeds proved to be a powerful tool in the authentication of their botanical origin, as well as for potential detection of berry-based products adulteration.