Abstract In Russia, live vaccine strains Brucella abortus 82 and B. abortus 75/79-AV have been widely and extensively utilized for specific prophylaxis of cattle brucellosis. To differentiate these vaccine strains from each other and laboratory collection of other vaccine ( n = 4), reference ( n = 15) and field Brucella strains ( n = 61), the multiple-locus variable-number tandem repeat (VNTR) analyses (MLVA) were used with 12 loci containing tandem repeats from 134 bp to 8 bp recently described for Brucella spp. The results obtained by MLVA typing revealed that the vaccines are genetically closely related. Three sufficiently stable in vitro VNTR loci were chosen in order to discriminate, in single tube Multiplex PCR, products of the vaccine strains from the other Brucella strains. Our results demonstrated that MLVA in Multiplex PCR format is a rapid, easy, economical and efficient tool for identification of vaccine strains and will be validated in future large scale typing of Brucella isolates circulating in Russia.