Objectives: The WHO European Region set targets for the control of hepatitis B through immunization, including prevalence of hepatitis B surface antigen (HBsAg) at ≤0.5% in vaccinated cohorts. The Republic of Moldova implemented universal hepatitis B vaccination since 1995. We conducted a nationwide representative serosurvey to estimate HBsAg seroprevalence in children born in 2013 to validate hepatitis B control targets. Methods: We used probability-based sampling and a two-stage cluster design. All children born in 2013 and registered in primary healthcare facilities were eligible for participation. We tested blood samples of all participants for hepatitis B core antibody (anti-HBc), using Enzyme-Linked Immunosorbent Assay (ELISA). Anti-HBc-positive samples were tested for HBsAg and HBsAg-positive samples confirmed, using ELISA. We obtained information on hepatitis B vaccination from vaccination cards. Results: Of 3352 sampled children, 3064 (91%) participated. Most participating children were 7 years old (n = 3030, 99%), 1426 (48%) were girls. The weighted, national seroprevalence estimate was 3.1% (95% confidence interval = 2.1-4.5) for anti-HBc and 0.21% (95% confidence interval = 0.08-0.53) for HBsAg. Conclusion: The study demonstrated the impact of hepatitis B vaccination and allowed the Republic of Moldova to validate regional hepatitis B control targets. Other countries with high vaccination coverage could use hepatitis B serosurveys and apply for validation. Sustained efforts in the Republic of Moldova will be crucial on the path to hepatitis B elimination.