Several molecular methods have been employed for Borrelia species identification. Newly developed technology, real-time polymerase chain reaction (RT-PCR), combines simultaneous amplification, detection and differentiation of strains in one PCR run. The aim of the study was to perform and evaluate RT-PCR for Borreliaburgdorferi sensu lato species identification. Borrelia species identification was accomplished on 374 Borrelia strains using two approaches: 1.) MluI restriction of entire borrelial chromosome (MluI-large restriction fragment patterns, LRFP), and 2.) RT-PCR targeting hbb gene and specific melting temperature (Tm) detection. The results of the two molecular methods were compared. With MluI-RFLP we were able to differentiate all Borrelia species and their subtypes within particular species. RT-PCR based on Tm determination identified unique strains within the species Borreliaafzelii (Tm 66.11 degrees C), B. burgdorferi sensu stricto (Tm 68.18 degrees C), Borreliaspielmanii (Tm 59.45 degrees C) and Borreliavalaisiana (Tm 59.62 degrees C). We were not able to distinguish the last two species that shared almost identical Tm. The large majority of Borreliagarinii strains shared Tm 51.42 degrees C, while subtype Mlg4 was characterized by Tm 56.87 degrees C. Strains of Borrelialusitaniae species also were heterogeneous; human isolate had Tm 63.47 degrees C while two tick isolates shared Tm 61.77 degrees C. Differences inside hbb gene enabled differentiation of the majority of Borrelia species, and revealed two clusters within B. garinii and B. lusitaniae species, respectively, but it was not possible to distinguish B. spielmanii form B. valaisiana. The major advantage of RT-PCR was that it was easy to perform and that the results were obtained within a few hours.