Staphylococcus aureusisolates from dairy cow mastitis are not always consistent with the characteristic morphology described, and molecular investigation is often needed. The aim of the study was to develop a duplex real-time PCR assay for rapid identification ofStaph. aureusisolates, targeting bothnucandSa442. Overall, 140 isolates collected from dairy cow mastitis in 90 different herds, were tested. All strains had been identified using morphological and biochemical characteristics. DNA from each strain was amplified in real-time PCR assay, to detectnucorSa442. Thereafter, a duplex real-time PCR assay was performed, and specificity of the amplified products was assessed by high resolution melting curve analysis. Out of 124Staph. aureusisolates, 33 did not show the typical morphology or enzymic activity; in 118 strains, the two melt-curve peaks consistent withnucandSa442were revealed, while 2 isolates showed only the peak consistent withSa442. Four isolates bacteriologically identified asStaph. aureus, were PCR-negative and were further identified asStaph. pseudintermediusby sequencing.Staph. pseudintermediusand coagulase-negative staphylococci did not carrynucorSa442. The results showed the correct identification of all isolates, comprehending also coagulase—or nuc-negativeStaph. aureus, while other coagulase-positive Staphylococci were correctly identified as non-Staph. aureus. Both sensitivity and specificity were 100%. High resolution melting analysis allowed easy detection of unspecific products. Finally, the duplex real-time PCR was applied directly to 40 milk samples, to detect infected mammary quarters. The assay confirmed the results of bacteriological analysis, onStaph. aureus-positive or—negative samples. Therefore, the proposed duplex real-time PCR could be used in laboratory routine as a cost-effective and powerful tool for high-throughput identification of atypicalStaph. aureusisolates causing dairy cow mastitis. Also, it could be applied directly to milk samples, to detectStaph. aureusmammary infections avoiding bacteriological analysis.