It is still under investigation, whether all Borrelia sp. causing Lyme borreliosis and other diseases are already identified and properly classified as human pathogens. For this reason, it is of great importance to develop a diagnostic ELISA test that detects all Borrelia sp. The aim of this study was to identify conserved DNA and protein regions present in all currently known Borrelia sp. In experimental studies 31 available Borrelia sp. genomes were aligned and screened for the presence of evolutionary conserved regions. As a result of bioinformatics analysis, one evolutionally conserved DNA region encoding a core fragment of the S21 ribosomal protein was identified. Both a couple of genus-specific PCR primers and the S21 protein B-cell epitope were designed for prospective diagnostic purposes.