The safeguard of river buffalo Mozzarella cheese, a Protected Designation of Origin dairy product, has prompted an analytical study to trace the milk and curd used as raw material in cheesemaking. This is to prevent the illegal use of milk or curd from different geographical areas outside of those indicated in the official production protocol. For this purpose, we studied primary proteolysis occurring in fresh and frozen milk and curd to identify a molecular marker that could indicate the raw material used. Whole casein from frozen river buffalo milk was separated using cation-exchange chromatography and sodium dodecyl sulfate-PAGE, and a protein component with an estimated molecular weight of 15.3 kDa was detected. This protein component was revealed in fresh river buffalo milk as a faint electrophoresis band, which drastically increased in intensity in refrigerated and frozen milk as well as in curd and was found to be associated with beta-CN through hydrophobic interaction. By using matrix-assisted laser desorption/ionization-time of flight peptide mass mapping, this component was identified as the C-terminal fragment f(69-209) of beta-CN (expected molecular weight of 15,748.8 Da). beta-Casein f(69-209), originating from the early hydrolysis of Lys(68)-Ser(69) by plasmin, has no counterpart in bovine milk. The increased rate of hydrolysis by plasmin toward the cleavage site Lys(68)-Ser(69) has to be ascribed to the elevated proline content of the peptide 61-73. The favored production of beta-CN f(69-209) has also drawn attention to the complementary proteose peptone beta-CN f(1-68) that is presumed to play a physiological role in inducing milk secretion similar to that of beta-CN f(1-29). The higher in vivo and in vitro production rate, compared with gamma(1)-CN formation, indicates that beta-CN f(69-209) and its complementary fragment are candidate molecular markers to evaluate milk and curd freshness. We suggested [corrected] indirect ELISA analysis based on the determination of remaining nonhydrolyzed beta-CN to perform a quantitative evaluation of proteolysis.