Abstract: A real-time polymerase chain reaction (PCR) assay was developed to detect a bovine-specific mitochondrial DNA sequence in `buffalo` Mozzarella cheese by using primers targeting the cytochrome oxidase subunit 1 (COI) gene. Hot-start PCR, primer design, annealing and signal acquisition temperatures were exploited to obtain reliable analytical conditions which yielded a 134-bp amplicon from cow`s DNA only. Water buffalo`s DNA didn`t originate any amplification product. DNA isolated from blood was used to test primers` specificity and to construct a calibration curve in order to quantify bovine DNA concentration in governing liquid. The method was capable to detect low amounts of cow`s DNA in governing liquid samples. Due to low detection limit and fast, simple execution, the analytical protocol described in this work is suitable to become a common tool to detect the fraudulent addition of cow milk in water buffalo Mozzarella cheese.