Brucellosis is one of the world's major zoonoses that still has veterinary, public health and economic concern in many parts of the world. In livestock, brucellosis is the major impediment for trade and export. In this work a total of 118 different samples (45 serum samples, 45 blood samples, stomach contents of 18 aborted feati and 10 fecal samples) were collected from 45 diseased and apparently healthy cows at selected veterinary clinics at El-Sharkia governorate. The serum samples were subjected to investigation by different serological methods (Rose Bengal, BAPAT, CFT and ELISA) for detection of bovine brucellosis. While, molecular differentiation between Brucella and Yersenia enterocolitica O:9 were made on blood samples, stomach contents and  fecal samples of sero positive animals for brucellosis using multiplex PCR assay. The results revealed that 26 out of 45 serum samples were positive for brucellosis, as detected by ELISA while 19 samples gave positive amplification of 223bp for Brucella and seven positive amplification of 325bp for Yersinia representing 26.92% of total brucellosis seropositives of which two samples (7.69%) were positive for both Yersinia and Brucella as tested by multiplex PCR assay. Therefore, we concluded that multiplex PCR proved to be a reliable molecular method for differentiation between Brucella and Yersinia as rapid diagnostic tool directly from clinical specimens.