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• The first comprehensive molecular typing of Brucella spp. infecting humans and livestock in Iran is presented. • Twenty –three Brucella isolates were characterized by phenotypic typing, AMOS-PCR, Bruce-ladder and MLST. • B. melitensis isolates belonged to 3 MLST-9 genotypes ST8, ST7, and ST102 – the latter previously reported only once, from a traveler with links to Iran. • Two widely dispersed MLST-9 genotypes were identified from B. abortus isolates (ST1 and ST2). • The study provides the first data describing how Brucella isolates from Iran relate to the global population structure. Brucellosis is an endemic infection in Iran and represents a serious health problem in humans and livestock causing important economic losses. The objective of this study was to undertake molecular characterization of Brucella spp. isolated from humans and livestock in several provinces of Iran including by multi-locus sequence typing (MLST), in order to understand the genotypes circulating in Iran and their relationship to genotypes globally. A total of 23 Brucella isolates were isolated from eight milk samples (seven cows, and one camel), human blood samples (seven), bovine lymph nodes (two), and samples from aborted fetuses (three sheep, two cows, and one goat). Phenotypic and molecular identification of Brucella isolates was performed on all isolated bacteria and showed that all were either Brucella melitensis or Brucella abortus. B. melitensis was associated with ovine/caprine and camel samples, most human isolates, and a significant minority of cattle isolates. In contrast B. abortus from livestock was associated only with isolations from bovine samples, as well as a single human sample. These results indicate that both B. melitensis and B. abortus contribute to the human brucellosis burden in Iran. B. melitensis isolates comprised three MLST-9 genotypes, the common and globally distributed ST8, a single representative of ST7, and several additional examples of ST102, a genotype previously only reported in a single isolate from a human brucellosis case believed to be acquired through travel to Iran. B. abortus isolates represented two globally common MLST-9 genotypes (ST1 and ST2), with relationships to biotype and other PCR-based typing methods consistent with previous observations. The results provide the basis for further studies examining the molecular epidemiology of Brucella circulating in Iran and the relationships of local isolates to those present globally. [ABSTRACT FROM AUTHOR]