The main object of the study is to demonstrate that Bruce-ladder multiplex PCR, which is capable of identify all Brucella species including vaccinal strains, is a safe and practical method that is alternative to bacteriological culture methods. Brucellosis is a zoonosis of great socio-economic importance which is endemic in Turkey. In three-year period from 2009 to 2011, a total of 281 Brucella spp. isolates from cattle, sheep and goats were identified and characterized by both conventional biotyping procedures and the PCR based method called Bruce-ladder. Species identification from both phenotypic testing and Bruce-ladder was concordant for all isolates tested. The results showed preferred host pattern in B.abortus isolates. In terms of species identity, it was found that 94% of the isolations derived from cattle were B.abortus and were exclusively biotype 3. B.melitensis biotype 3 and biotype 1 were the prevelant biotypes in sheep and goats. Two isolates from sheep were identified as B.melitensis Rev.1 vaccine strain. Surprisingly, one of the goat isolates was identified as B.abortus S19 vaccine strain. Other interesting observations were the isolation of penicilin-sensitive non vaccine strains of B.abortus and B.melitensis as well as an isolation of an atypical B.abortus biotype 3 strain on the basis of growth inhibition in the presence of basic fuchsin dye. At the end of this study, it was concluded that monitoring Brucella isolates and identify them are indispensable epidemiological tool for brucellosis control