Background: Tick-borne encephalitis (TBE) is a disease with mandatory declaration in the EU since 2012. Information regarding the seroprevalence of the disease across Romania is limited, and only sporadic cases are rarely reported. We aimed to identify new areas of TBEV infection in different counties of Romania. Methods: We conducted a serosurvey assessing the immune response to TBEV infection in adult populations from rural areas in different counties of the country. Seropositivity was defined by a positive TBEV IgM/IgG ELISA test and confirmed by serum neutralization. Results: We collected 1116 samples from 15 different localities in 10 counties (divided into endemic/border/non-endemic counties) across Romania. Overall, 26 (2.3%) of the samples were tested positive using the TBEV ELISA assay in six counties. In those counties, 3.7% of sera were positive, varying from 1.4% to 6.9% per county. After performing the neutralization assay, seven (0.6%) samples were confirmed positive, interestingly all from one site in Sibiu County, where the seroprevalence was 9.7%. Conclusions: The identification of positive serum samples in serosurveys appears to rely on the discovery of TBEV microfoci. Further serological surveys should be conducted in Romania, especially after the identification of positive TBEV patients presenting for medical care.