Aim To primarily estimate the sensitivity (Se) and specificity (Sp) of the commercially available Mastit4 quantitative PCR (qPCR) assay and bacterial culture (BC) for diagnosis of intramammary infections (IMI) and teat apex colonization (TAC) with coagulase-negative staphylococci (CNS) at different cut-offs for qPCR cycle threshold values using Bayesian latent class analysis. A secondary objective was to evaluate two cut-offs of BC for diagnosis of IMI and TAC with CNS. Methods and Results We randomly selected 13-20 cows with subclinical mastitis from eight dairy herds. Teat skin samples and aseptically collected foremilk samples were collected from the right hindquarters (n = 149) for BC and qPCR analysis. The Se of qPCR was always higher than BC(Se )in diagnosis of IMI, however; the Sp of BC was higher than qPCR(Sp). BC(Se )and BCSp showed no substantial difference between the tested BC cut-offs. In contrast to IMI, estimates of BC and qPCR in diagnosing TAC were different. BCSe was higher than qPCR(Se) at all tested cut-offs, however; qPCR(Sp) was higher than BCSp. Conclusion The overall performance of qPCR is higher than BC in the diagnosis of IMI; however, the performance of BC is better than qPCR in diagnosis of TAC. The qPCR and BC are valid diagnostics for bovine IMI with CNS. However, for TAC, both techniques require further investigation to reduce the uncertainty of the true status of the quarter and teat skin. Significance and Impact of the Study We reported, for the first time, the diagnostic performance of new mastitis technology (Mastit4 PCR) and culture for detection of CNS in milk and nonmilk samples in dairy herds with automatic milking systems. Our findings will improve the interpretation of the test results of culture and qPCR assay and subsequently, will strengthen the control of IMI with CNS in dairy cows.