BackgroundTick-borne encephalitis (TBE) can be a severe neurological disease. Identifying ecological factors that may facilitate tick-borne encephalitis virus (TBEV) circulation in the Netherlands could improve awareness and detection.AimWe aimed to identify ecological factors affecting TBEV circulation in the Netherlands and to determine if there is sustained circulation and spread of the virus.MethodsBetween June and September 2021, rodents and ticks from three previously TBEV-positive locations were tested for TBEV by PCR. We sequenced TBEV and compared the sequences with previous and subsequent sequences from the Netherlands and other countries to investigate the spread of TBEV-variants.ResultsWe captured 383 rodents, 928 feeding ticks and 1,571 questing