Tick-borne encephalitis (TBE) is an infectious disease of the central nervous system. The causative agent is the tick-borne encephalitis virus (TBEV), which is most commonly transmitted by tick bites, but which may also be transmitted through the consumption of raw dairy products or, in rare instances, via infected transfusions, transplants, or the slaughter of infected animals. The only effective preventive option is active immunization. Currently, two vaccines are available in Europe—Encepur®and FSME-IMMUN®. In Central, Eastern, and Northern Europe, isolated TBEV genotypes belong mainly to the European subtype (TBEV-EU). In this study, we investigated the ability of these two vaccines to induce neutralizing antibodies against a panel of diverse natural TBEV-EU isolates from TBE-endemic areas in southern Germany and in regions of neighboring countries. Sera of 33 donors vaccinated with either FSME-IMMUN®, Encepur®, or a mixture of both were tested against 16 TBEV-EU strains. Phylogenetic analysis of the TBEV-EU genomes revealed substantial genetic diversity and ancestry of the identified 13 genotypic clades. Although all sera were able to neutralize the TBEV-EU strains, there were significant differences among the various vaccination groups. The neutralization assays revealed that the vaccination using the two different vaccine brands significantly increased neutralization titers, decreased intra-serum variance, and reduced the inter-virus variation.