At least three Borrelia species (Borrelia afzelii, Borrelia garinii and Borrelia burgdorferi sensu stricto) cause disease in humans, but Borrelia spielmanii, Borrelia valaisiana, Borrelia lusitaniae and Borrelia bissettii have also been reported to be rare or potential causes of human disease in Europe. Pulsed-field gel electrophoresis after MluI restriction of the genomic DNA (MluI large restriction fragment patterns, LRFPs) represents one of several approaches that have been used to assess Borrelia genotypic characteristics. The aim of the present report was to analyze the value of MluI-LRFP for identification of B. burgdorferi sensu lato at a species level and for further species subtype delineation. Results of the present study are based on 1487 B. afzelii strains, 285 B. garinii strains, 29 B. burgdorferi sensu stricto strains, 23 B. valaisiana strains, 8 B. spielmanii strains and 3 B. lusitaniae strains. Using MluI-LRFP, we were able to delineate all Borrelia species included in the study. Each of the six examined Borrelia species displayed unique MluI-LRFPs that enabled straightforward separation of strains into particular species, and also of strains within species. The subtypes of B. afzelii (Mla2 and Mla3), B. spielmanii (Mls1 and Mls2) and B. lusitaniae (Mll1 and Mll2) uncovered in the present analysis have not been reported previously. MluI-LRFP represents a highly specific and reproducible method for Borrelia identification.