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Background  A novel  Brucella  strain closely related to  Brucella  ( B .)  melitensis  biovar (bv) 3 was found in Croatian cattle during testing within a brucellosis eradication programme.    Case presentation  Standardised serological, brucellin skin test, bacteriological and molecular diagnostic screening for  Brucella  infection led to positive detection in one dairy cattle herd. Three isolates from that herd were identified to species level using the Bruce ladder method. Initially, two strains were typed as  B. melitensis  and one as  B. abortus , but multiplex PCR based on IS 711  and the Suis ladder showed that all of them to belong to  B. melitensis,  and the combination of whole-genome and multi-locus sequencing as well as Multi-Locus Variable numbers of tandem repeats Analysis (MLVA) highlighted a strong proximity within the phylogenetic branch of  B. melitensis  strains previously isolated from Croatia, Albania, Kosovo and Bosnia and Herzegovina. Two isolates were determined to be  B. melitensis  bv. 3, while the third showed a unique phylogenetic profile, growth profile on dyes and bacteriophage typing results. This isolate contained the 609-bp  omp31  sequence, but not the 723-bp  omp31  sequence present in the two isolates of  B. melitensis  bv. 3.    Conclusions  Identification of a novel  Brucella  variant in this geographic region is predictable given the historic endemicity of brucellosis. The emergence of a new variant may reflect a combination of high prevalence among domestic ruminants and humans as well as weak eradication strategies. The zoonotic potential, reservoirs and transmission pathways of this and other  Brucella  variants should be explored.    Supplementary Information  The online version contains supplementary material available at 10.1186/s12917-021-02833-w.

New Brucella variant isolated from Croatian cattle