It has been suggested that canine intestinal spirochaetes consist of Brachyspira pilosicoli and a group of strains that has been provisionally designated 'Brachyspira canis'. The purpose of the present study was to compare 22 spirochaete isolates that were obtained from intestinal specimens of dogs in Sweden (n = 12), Norway (n = 4), the United States (n = 3), Australia (n = 2) and Germany (n = 1) with type and reference strains, as well as field isolates, of Brachyspira species by five biochemical tests and determination of almost-complete 16S rDNA sequences. In an evolutionary tree derived from 16S rDNA sequences, the canine isolates grouped into three clusters. One cluster included the type strain of porcine B. pilosicoli, whereas a second larger cluster, which was monophyletic, contained a canine strain that was identified previously as 'B. canis'. The third cluster consisted of three canine isolates of Scandinavian origin, which grouped together with the type strain of the species Brachyspira alvinipulli (pathogenic to chicken). These three genotypes, which were identified on the basis of 16S rDNA sequences, corresponded to four phenotypic groups based on biochemical testing. Two biochemical tests, hippurate hydrolysis and alpha-galactosidase production, were sufficient for rapid identification of each canine cluster.