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Lyme borreliosis (LB) is the most frequent vectorborne disease in the Northern Hemisphere. It is a complex bacterial zoonosis involving vertebrate hosts and hard ticks of the genus Ixodes. The causative agents, bacteria of the LB group of spirochaetes, form a species complex comprising 17 named species. As is the case for most microbial pathogens, epidemiological and ecological studies require appropriate genotyping. Although the use of single loci may provide rapid results, there are serious disadvantages, in particular when inferring evolutionary relationships or geographic population structure. A novel multilocus sequence analysis (MLSA) system of the LB group spirochaetes has been developed based on housekeeping genes to overcome these problems. Here, the system is optimized and tested using extracted spirochaetal DNA directly from ticks and then utilized to obtain insights into the migration and spread of individual species as well as to investigate the evolutionary origins of the species complex. Species belonging to the LB group of spirochetes display different patterns and levels of host specialisation which makes this an ideal system to study the impact of host associations on spread of zoonotic tickborne diseases. For example, Borrelia valaisiana and B. garinii are transmitted exclusively by birds while B. afzelii is transmitted by rodents. I demonstrate that the migration of the LB species is dependent on, and limited by, the migration of their respective hosts. I also show the presence of B. afzelii strains in England and, through the use of the MLSA scheme, demonstrate that the strains are highly structured. A close evolutionary relationship between B. afzelii and its rodent host species is shown. Furthermore, through phylogenetic analyses, some evidence of a coevolutionary relationship between the LB group species and their major group of vector species, the Ixodes persulcatus species complex, is presented and a Eurasian origin for the species group is suggested.