Tick-borne encephalitis (TBE) is a member of the Flaviviridae family. Strong cross-reactions can occur between members of this family, so that it may be difficult to diagnose specific flavivirus infections, especially when tests with frequent cross-reactions e.g. ELISA tests are used. We tested 238 sera with borderline titers for TBE using the indirect immunofluorescence or neutralization test for other flaviviruses (yellow fever, dengue, West Nile) to detect cross-reactions due to other flavivirus infections or flavivirus vaccination. Only one serum reacted against all the flaviviruses tested, indicating cross-reactivity due to infection with any of the flaviviruses. Two other sera exhibited low antibody titers against yellow fever, which could be confirmed by the neutralization test, indicating recent yellow fever vaccination. None of the other sera reacted at all against any of the flaviviruses tested in the tests used, which indicates false positive reactions with the TBE-ELISA. Sera with borderline titers in the TBE-ELISA in particular should be retested using other test systems (preferably neutralization) and for other flaviviruses (yellow fever, dengue, West Nile) to detect cross-reactions and to confirm positive results.