Abstract: A mutiplex polymerase chain reaction(PCR) assay was developed for the simultaneous detection of the four major agents of bovine mastitis, Staphycococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae and yeast. The target sequences of Staphycococcus aureus, Streptococcus agalactiae,and Streptococcus dysgalactiae were the 16S to 23S rRNA spacer regions. The target sequence of yeast was 18S rRNA region. The results showed that the assay was specific. Simulation bacteria infection samples were made according to Skladny’s method. The sensitivity of the mutiplex PCR was lower in detecting Staphycococcus aureus, Streptococcus dysgalactiae and yeast of the milk. The sensitivity of mutiplex PCR in detecting Staphycococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae and yeasts was 104CFU/mL，102CFU/mL，103CFU/mL and 103CFU/mL respectively.The performance of the mutiplex PCR and traditional culture method were evaluated by examining 46 clinical mastitis milk samples and 167 subclinical mastitis milk samples. The results showed that mutiplex PCR was more sensitive than culture in detecting Staphycococcus aureus and yeasts(P<0.01), but was not different from the culture method in detecting Streptococcus agalactiae and Streptococcus dysgalactiae (P>0.05).