The manufacturing of Buffalo Mozzarella PDO (Protected Designation of Origin) cheese requires the exclusive use of fresh buffalo milk, which must be transformed into cheese within 60 hours after milking. The limited availability of buffalo milk and simultaneous increase in Mozzarella demand during the summer cause producers to use frozen intermediates (milk and/or curd) in the cheese-making process. These practices are not allowed. Few data are available in the literature about the effects of freezing on buffalo milk and curd. Recent studies demonstrated that the use of frozen buffalo milk can be detected in mozzarella cheese based on the increase in casein fragment γ4-CN. This work aims to verify the possibility of tracing the presence of frozen curd in Buffalo Mozzarella PDO cheese. The electrophoresis technique was used to reveal the presence of γ4-CN. Equivalent concentrations of this fragment were found in fresh and frozen curd that were stored for 9 months. Our results suggest that γ4-CN cannot be used to discriminate fresh PDO Mozzarella and Mozzarella cheese produced from frozen curd. A second objective of the work was to evaluate the effects of freezing on curd lipids. In particular, the fatty acid and mono-diglyceride profiles were evaluated. Significant differences were found in the amounts of 1,2-Dipalmitin and 1,3-Diolein between fresh curd and curd that was stored for 9 months at freezing temperatures. Although some significant differences were found in the mono-diglyceride profiles, no objective marker that can distinguish between fresh and frozen products is currently available.