AbstractBrucellosis is one of the most common bacterial zoonoses worldwide affecting not only livestock and wildlife but also pets. Canine brucellosis is characterized by reproductive failure in dogs. Human Brucella canis infections are rarely reported but probably underestimated due to insufficient diagnostic surveillance. To improve diagnostics, we investigated dogs in a breeding kennel that showed clinical manifestations of brucellosis and revealed positive blood cultures. As an alternative to the time-consuming and hazardous classical identification procedures, a newly developed species-specific intact-cell matrix-assisted laser desorption/ionization–time of flight mass spectrometry analysis was applied, which allowed for rapid identification of B. canis and differentiation from closely related B. suis biovar 1. High-throughput sequencing and comparative genomics using single nucleotide polymorphism analysis clustered our isolates together with canine and human strains from various Central and South American countries in a distinct sub-lineage. Hence, molecular epidemiology clearly defined the outbreak cluster and demonstrated the endemic situation in South America. Our study illustrates that MALDI-TOF MS analysis using a validated in-house reference database facilitates rapid B. canis identification at species level. Additional whole genome sequencing provides more detailed outbreak information and leads to a deeper understanding of the epidemiology of canine brucellosis.