Accurate and precise differentiation of staphylococci isolated from milk is of importance for udder health management. In particular, the rapid and specific identification ofStaphylococcus aureusplays an essential role in the prevention and treatment programs for bovine mastitis. Plasma gelatinization in coagulase assays is routinely used to discriminateS. aureusfrom other species by detecting the presence of extracellular free staphylocoagulase. However, rarely occurring coagulase-deficientS. aureusstrains can be responsible for clinical and subclinical mastitis cases. By investigatingS. aureusisolates from a single herd over a 10-year period we identified the persistence of a phenotypically coagulase-negativeS. aureusstrain and pinpointed the possible cause to a single base pair deletion in thecoagene sequence. Our results support the need to integrate primary biochemical tests with molecular/sequence analysis approaches for correctly identifying and discriminating atypicalS. aureusin bovine herds, as the coagulase test alone may fail to detect persistent mastitis-causing strains.