This study compared the fatty-acid profiles of Brucella canis blood culture isolates obtained from infected dogs in the UK, Germany, Japan, South Africa, Peru, Mexico, Colombia, and Argentina, and from a human clinical case in Argentina, to a bank of isolates obtained from canine outbreaks in the USA. Analysis of a total of 42 B. canis isolates and one reference strain found a marked variation within the species. Fatty-acid analysis showed that only the isolates from Argentina, Colombia, and Mexico, which included the human B. canis isolate, contained a specific fatty acid, 19:0 cyclopropane (lactobacillic acid), w8c (cis-11,12-methylene octadecanoic acid), and that this fatty acid, when present, made up a large percentage of overall fatty-acid content. Prior to this study, the cellular fatty-acid 19:0 cyclopropane had been identified in all of the species of Brucella considered to be pathogenic to humans (B. abortus, B. melitensis, B. suis) except for B. canis. Discovering that this fatty acid not only occurs in B. canis, but also that it is only present in some strains of the species provides a new focus for investigations aimed at identifying the cause of reported geographical variability in human B. canis infection, and at finding predictors of biological behaviour and human pathogenicity within this Brucella species.