Tick-borne encephalitis (TBE) is an arthropod-borne virus disease with more than 10,000 cases annually in Europe and Asia. Vaccines have been developed to help protect against TBE. Pre-requisite for consistent production of a viral vaccine is the stable replication of the virus from early development steps to the end of production. We have analysed the nucleotide sequence encoding the surface glycoprotein E of TBE virus strain K23 derived from the virus master seed bank, the working seed bank and a production lot and no sequence variation was found in these three virus samples. We conclude that the genetic constitution during the production process of the analysed TBE vaccine is highly consistent and stable, which is a requirement for defined antigen production process.