Relapsing fever Borrelia challenge microbiological typing as, unlike other microbes, they possess segmented genomes maintaining essential genes on large linear plasmids. Antigenic variation further complicates typing. Intragenic spacer (IGS, between 16S-23S genes), provides resolution among Lyme-associated and some relapsing fever spirochaetes. When applied to East African relapsing fever borreliae, two and four types were found respectively among Borrelia recurrentis and B. duttonii. However, IGS typing was unable to discriminate between the tick- and louse-borne forms of disease, raising the question as to whether these are indeed separate species. To address this question, further genes were sequenced to produce a multi-locus approach to resolve whether these are either a single or different species. Various housekeeping genes were selected from data deposited for B. hermsii (limited sequence information exists for either B. recurrentis or B. duttonii). Of selected targets, sufficient data was produced only from glpQ. Further genes analysed included flaB, rrs rDNA, and P66 outer membrane protein. Sequence comparison of multiple genes was undertaken, but restricted through the limited number of available isolates of these notoriously fastidious organisms that until recently were considered non-cultivable. Whereas the IGS typing was applied to a range of clinical isolates, patient blood samples and arthropod vectors, other genes were sequenced only from cultivable strains, potentially introducing a bias to the results. Our data highlights the remarkable similarity between these Borrelia with only minor differences at the nucleotide level. Collectively, this suggests a common ancestral lineage for these spirochaetes, with the limited differences revealed at the nucleotide level from these cultivable strains being able to divide both “species” into separate clades; however, it must be stressed that these differences ranged from 2 to 10 nucleotides depending on the gene used. It is more likely that these are clades of the same species, which have accumulated adaptive changes through time and pressures of different vector transmission. In contrast, the IGS sequence, being non-coding, is not under such selection and in consequence probably reflects changes accumulated over time alone, but without the constraints of producing functional gene products. Full genomic sequence analysis should reveal further insights into the taxonomic relationship between these microbes and elucidate the molecular basis of arthropod competence and pathogenicity among these spirochaetes.