Aim of this study is to apply a population proteomic approach for the characterization of local chicken breeds. The experiment involved a total of 29 males of Pépoi, Padovana, and Ermellinata Italian local chicken breeds. Sarcoplasmic protein fractions of breast muscle were analysed by two-dimensional electrophoresis. Image analysis followed by statistical analysis enabled to differentiate groups of individuals based on similarities of protein expression. Individuals were distinguished into clusters and groups, corresponding to the breed of origin. Distances among individuals calculated using data on spot volumes were used to draw a neighbour joining tree, showing clear breed differentiation ; 10 of these were identifie by Mass Spectrometry revealing preliminary evidences on the mechanics of the breed differentiation process. Results evidenced the good performances of proteomic applied to breed characterization studies.