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Background and Aims: Brucellosis is an infectious zoonotic disease responsible for reproductive disorders among animals and significant economic losses to the meat and milk supplychain. The purpose of this study was therefore to evaluate and diagnose the species and strains of Brucella isolates among cattles via multiplex polymerase chain reaction. Materials and Methods: In this study, blood samples were taken from 22 cows diagnosed as having brucellosis by PCR test. Blood samples were cultured in BACTEC vials and incubated at 37 degrees Celsius for 5 days following which each sample was cultured on Brucella agar and kept for 3 days under Co2 condition.The 16SrRNA gene was used as the target for the detection of Brucella genus and the bcsp31 gene was used for the detection of Brucella biovars. The PCR products were electrophoresed on %1 agarose and compared with standard strains. Results: The PCR results showed a total of 22 positive samples for Brucella among cattles. Out of the 22 cattle positive samples, 19 were identified as B.abortus (biovars 1, 2, 3, 4, 5) and 3 as B. melitensis (biovars 1, 2, 3). Conclusions: Brucella abortus biovars 1 and 3 and B .melitensis biovar 1 are the most prevalent biovars among cattles in Lorestan province, Iran. Moreover, multiplex PCR method could be used to improve the qualitative level and the Brucella diagnosis speed in laboratories. [ABSTRACT FROM AUTHOR]