SDS-PAGE of extracted surface-associated proteins of Lactobacillus rhamnosus strains E/N, Oxy, and Pen, was performed. The obtained protein patterns allowed differentiation of the examined strains, which was not accomplished by the commonly used RAPD genotypic method. The differentiation by the SDS-PAGE method proved to be a useful tool for strain-specific identification, which was further confirmed by 2DE analysis. Therefore, it can be used as an alternative or complementary method for both conventional and genotypic identification procedures, especially when closely related lactobacilli isolates are identified.