MultiLocus sequence typing (MLST) is considered a powerful method to unveil relationships within bacterial populations and it constitutes an economical and fast alternative to whole genome sequencing. We used this method to understand whether there are differences in human pathogenicity within and between different Borrelia burgdorferi sensu lato species. Therefore, 136 strains from human patients or ticks from Europe were included in MLST analyses. The scheme employed used eight chromosomally located housekeeping genes (i.e. clpA, clpX, nifS, pepX, pyrG, recG, rplB and uvrA). We investigated Borrelia afzelii, one of the predominant species in Europe, and B. burgdorferi sensu stricto (s.s.), because it allowed comparative analysis to strains from the USA. We typed 113 patient isolates as well as 23 tick isolates. For further comparative purposes an additional 746 strains from Europe and the USA were included from the MLST website http://borrelia.mlst.net. We observed an overlap of the B. burgdorferi s.s. populations from Europe and the USA isolated from human patients while there was no overlap of the populations found in tick vectors. Further results indicate that B. afzelii was significantly less associated with disseminated infection than B. burgdorferi s.s. and that B. burgdorferi s.s. from Europe caused neuroborreliosis to a significantly greater extent than B. afzelii or B. burgdorferi s.s. in the USA. Our data suggest that there may be an evolutionary basis of differential interspecies pathogenicity in Borrelia. This was not evident within Borrelia species: we found the same sequence types in patients with disseminated or localized symptoms when the number of strains was sufficiently high. We hypothesize that the finding that B. burgdorferi s.s. in Europe is much more associated with neuroborreliosis than in the USA maybe linked to factor(s) related to the human host, the tick vector or the bacterium itself (e.g. plasmid content and structure).