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• Canine brucellosis seems to be a frequent infection in Costa Rica. • Brucella canis was cultured from brucellosis outbreaks in kennel facilities and pet dogs in the Central Valley of Costa Rica. • Infections in kennel facilities were traced to stud dogs imported from Panama and Mexico. • Multiple locus variable number tandem repeats analysis (MLVA-16) and whole-genome sequencing (WGS) studies showed that B. canis Costa Rican strains comprise several lineages. • The tree topologies obtained by WGS and MLVA-16 just partially agreed, indicating that the latter analysis is not suitable for phylogenetic studies. • The fatty acid analysis demonstrates that CR phylogenetic lineages I and II lack lactobacillic acid, which correlates with a no functional cyclopropane synthase gene, while lineage III contains lactobacillic acid. • B. canis displaying putative functional cyclopropane synthase are phylogenetically intertwined with B. canis with non-functional protein, indicating that mutations have occurred independently in the various lineages. Brucellosis is a prevalent disease in Costa Rica (CR), with an increasing number of human infections. Close to half of homes in CR have one or more dogs, corresponding to ∼1.4 million canines, most of them in the Central Valley within or near the cities of San José, Heredia, and Alajuela. From 302 dog sera collected from this region, 19 were positive for Brucella canis antigens, and five had antibodies against smooth lipopolysaccharide, suggesting infections by both B. canis and other Brucella species. B. canis strains were isolated in the Central Valley from 26 kennel dogs and three pet dogs, all displaying clinical signs of canine brucellosis. We detected three recent introductions of different B. canis strains in kennels: two traced from Mexico and one from Panama. Multiple locus-variable number tandem repeats (MLVA-16) and whole-genome sequencing (WGSA) analyses showed that B. canis CR strains comprise three main lineages. The tree topologies obtained by WGSA and MLVA-16 just partially agreed, indicating that the latter analysis is not suitable for phylogenetic studies. The fatty acid methyl ester analysis resolved five different B. canis groups, showing less resolution power than the MLVA-16 and WGSA. Lactobacillic acid was absent in linages I and II but present in linage III, supporting the recent introductions of B. canis strains from Mexico. B. canis displaying putative functional cyclopropane synthase for the synthesis of lactobacillic acid are phylogenetically intertwined with B. canis with non-functional protein, indicating that mutations have occurred independently in the various lineages. [ABSTRACT FROM AUTHOR]