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A study on casein genetic polymorphism in water buffalo milk of Mediterranean breed, reared in Southern Italy, was carried out by means of immunoelectrophoretic and chromatographic techniques coupled with mass spectrometry analysis. When compared with reference α_s1-CN variant A, the results showed the occurrence of a new α_s1-casein (α_s1-CN) B genetic variant having a single silent amino acid substitution Leu¹⁷⁸(A)→Ser¹⁷⁸(B). In Mediterranean buffalo milks the allelic frequency of new α_s1-CN B was very similar to that determined in Mozzarella di bufala campana (MBC), a Protected Denomination of Origin (PDO) cheese, made exclusively from whole raw buffalo milk of this breed. The relative percentage of the two α_s1-CN variants, either in bulk milk or MBC cheese, can be used in defining the authenticity of PDO MBC cheese.