The incidence of tick-borne encephalitis (TBE) in Scandinavia is increasing and spreading geographically. Following two clinical cases of TBE hospitalised after tick bites in northern Zealand, Denmark, specific IgM and IgG antibodies against tick-borne encephalitis virus (TBEV) were demonstrated in acute serum samples of these patients. TBEV was identified by RT-PCR in ticks collected from the same location. This is the first report of TBEV in Ixodes ricinus leading to clinical cases in Denmark outside of Bornholm island.