Bovine mastitis is the greatest source of economic loss in the dairy industry. Rapid and definitive detection of causative agent is very important for treatment and control of the disease. The aim of this study was to compare culture and polymerase chain reaction techniques for diagnosis of agents in subclinical bovine mastitis. For this purpose, after conducting the California Mastitis Test on 540 cows, 79 milk samples were analysed by the classical culture method and simplex polymerase chain reaction. Forty-three of samples were found positive by both methods, differences were found only seven samples. While coagulase negative staphylococci these seven samples were determined by culture method, coagulase negative staphylococci and S. dysgalactiae were determined together by polymerase chain reaction. The results of this study indicate that the polymerase chain reaction is more sensitive than culture method and could detect pathogens at the species level within a few hours from directly milk samples. Rapid and relieable molecular techniques can be useful method in farm level detection for fast decision about the culling or treatment.