Bacteriological culture as a diagnostic tool for chronic infections with Staphylococcus aureus intramammary infection is not completely satisfactory. The cyclical shedding pattern of Staph. aureus with intervals of low excretion is considered to be the main reason. We recently developed a novel assay for Staph. aureus in milk, based on real-time quantitative PCR (QPCR). In a longitudinal study of chronically infected cows we evaluated the diagnostic properties of this test under field conditions. Diagnostic sensitivity of the novel test proved to be very high with a value of 99.4%; diagnostic specificity was 97.1%. In addition, the shedding patterns of Staph. aureus for the sampling period were analyzed. Using log(10)-transformed QPCR data and plotting them across sampling time revealed a sinusoidal shedding pattern in 6 of 11 naturally infected quarters. Shedding patterns obtained by QPCR and by bacteriological culture were synchronous. In conclusion, the novel test has a very high diagnostic sensitivity and specificity so that quarters chronically infected with Staph. aureus are reliably detected, independent from their actual shedding quantity.