The Danish island of Bornholm in the Baltic Sea has been known as a tick-borne encephalitis (TBE) natural focus for more than 60 years. TBE in humans is diagnosed on a regular basis either in inhabitants or tourists of the island. Other areas in Denmark have been suggested as possible risk areas of TBE. Despite the long-known endemicity on Bornholm and the possibility of the virus circulating in other areas, no data on the prevalences of TBE virus (TBEV) in ticks, or adequate molecular characterization and phylogenetic studies are available for the circulating TBEV strains. This study aimed to detect TBEV in ticks collected on the island of Bornholm and other possible risk areas, with the attempt to isolate the circulating viruses for molecular and phylogenetic analysis and confirm the presence of virus in the predicted risk areas. From 2014 to 2016, 9321 I. ricinus (nymphs, females, and males) were collected by flagging 31 locations in Denmark. The ticks were pooled and tested for TBEV by qPCR. The envelope gene of the detected TBE virus strains was amplified and sequenced by RT-PCR. After successful virus isolation, whole genome sequencing was performed. Phylogenetic analysis of the obtained sequences was done by the Maximum Likelihood method. One pool of 11 females and one pool of eight males from a total of 34 tick pools collected from the northwestern shore of lake Rubinsøen on Bornholm tested positive, resulting in a local estimated point prevalence of 0.6% [CI95% 0,1–1.85%] in this microfocus. We were not successful in confirming any other of the predicted TBEV-endemic areas. Alignment of the two complete E genes from Bornholm revealed identical sequences. Virus isolation and whole genome sequencing were succeeded from one of the positive samples. Phylogenetic analysis showed that the isolated virus had the closest phylogenetic relationship to TBEV sequences detected in Eastern and Central Europe.