Tick-borne encephalitis virus (TBEV), like other arthropod-transmitted viruses, depends on specific vectors to complete its enzootic cycle. It has been long known that Ixodes ricinus ticks constitute the main vector for TBEV in Europe. In contrast to the wide distribution of the TBEV vector, the occurrence of TBEV transmission is focal and often restricted to a small parcel of land, whereas surrounding areas with seemingly similar habitat parameters are free of TBEV. Thus, the question arises which factors shape this focal distribution of TBEV in the natural habitat. To shed light on factors driving TBEV-focus formation, we used tick populations from two TBEV-foci in Lower Saxony and two TBEV-foci from Bavaria with their respective virus isolates as a showcase to analyze the impact of specific virus isolate-tick population relationships. Using artificial blood feeding and field-collected nymphal ticks as experimental means, our investigation showed that the probability of getting infected with the synonymous TBEV isolate as compared to the nonsynonymous TBEV isolate was elevated but significantly higher only in one of the four TBEV foci. More obviously, median viral RNA copy numbers were significantly higher in the synonymous virus–tick population pairings. These findings may present a hint for a coevolutionary adaptation of virus and tick populations.