Danish farmers can order a real-time PCR mastitis diagnostic test on routinely takencow-level samples from milk recordings. Validation of its performance in comparison toconventional mastitis diagnostics under field conditions is essential for efficient control ofintramammary infections (IMI) with Staphylococcus aureus (S. aureus). Therefore, the objec-tive of this study was to estimate the sensitivity (Se) and specificity (Sp) of real-time PCR,bacterial culture (BC) and California mastitis test (CMT) for the diagnosis of the naturallyoccurring IMI with S. aureus in routinely collected milk samples using latent class anal-ysis (LCA) to avoid the assumption of a perfect reference test. Using systematic randomsampling, a total of 609 lactating dairy cows were selected from 6 dairy herds with bulktank milk PCR cycle threshold (Ct) value ≤39 for S. aureus. At routine milk recordings, auto-matically obtained cow-level (composite) milk samples were analyzed by PCR and at thesame milking, 2436 quarter milk samples were collected aseptically for BC and CMT. Resultsshowed that 140 cows (23%) were positive for S. aureus IMI by BC while 170 cows (28%)were positive by PCR. Estimates of Se and Sp for PCR were higher than test estimates of BCand CMT. SeCMTwas higher than SeBChowever, SpBCwas higher than SpCMT. SePCRwas 91%,while SeBCwas 53%, and SeCMTwas 61%. SpPCRwas 99%, while SpBCwas 89%, and SpCMTwas65%.In conclusion, PCR has a higher performance than the conventional diagnostic tests (BCand CMT) suggesting its usefulness as a routine test for accurate diagnosis of S. aureus IMIfrom dairy cows at routine milk recordings. The use of LCA provided estimates of the testcharacteristics for two currently diagnostic tests (BC, CMT) and a novel technique (real-time PCR) for diagnosing S. aureus IMI under field conditions at routine milk recordings inDenmark.