OBJECTIVE Staphylococcus argenteus is an emerging species of the Staphylococcus aureus complex. It has usually been misidentified as S. aureus by conventional methods and its characteristics. S. argenteus is potentially emerging in both humans and animals with an increasing global distribution. This study aimed to differentiate and identify S. argenteus from S. aureus collected and isolated from milk samples of subclinical bovine mastitis cases in Maha Sarakham Province, Northeastern Thailand. METHODS Forty-two isolates of S. aureus were studied from 132 individual milk samples collected from subclinical bovine mastitis cases of 15 dairy farms in three districts of Maha Sarakham, Thailand. The identification was confirmed by conventional and immune-agglutination methods. Fifteen representative isolates which were suspected as being S. argenteus were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). RESULTS The result from MALDI-TOF MS confirmed that seven from 15 isolates were S. argenteus and eight isolates were S. aureus. CONCLUSIONS This study indicated that MALDI-TOF MS used as an identification and classification method could accurately differentiate the novel species, S. argenteus, from the S. aureus complex which is usually misdiagnosed. In addition, the identification of S. argenteus seems to be very limited in technical difficulty despite the fact that it may be the important causative pathogen in bovine mastitis as well as a pathogenic bacterium in food and milk. Therefore, it is essential for both bovine medicine and veterinary public health to emphasize and recognize this bacterial pathogen as an emerging disease of staphylococcal bacteria that there is a need for further study of S. argenteus infections.