Background: Hepatitis B virus (HBV) infection is highly endemic in most of developing countries including Cambodia. This nationwide serosurvey was conducted to assess the impact of hepatitis B vaccination and to determine whether Cambodia met the WHO regional 2017 target of hepatitis B surface antigen (HBsAg) seroprevalence less than 1% in five-year-old children. Methods: A cross-sectional multi-stage cluster survey was conducted among children born during 2010-2012 and their mothers in Cambodia. HBsAg prevalence was estimated by rapid point-of-care testing, and demographic data, including vaccination history, were collected. Factors associated with children's failure to receive vaccination as well as HBsAg positivity were analysed by multivariate logistic analysis. Findings: Total of 2,520 children 5-7 year old and 2,028 mothers were recruited. In total, 78.4% of children received HepB birth-dose (HepB-BD); of these, 58.7% were administered ≤24 hours. Independent risk factors for children's failure to receive HepB-BD included children born at home, low maternal educational level and mothers' lack of knowledge about HBV. Overall HBsAg seroprevalence was 4.39% (3.50%-5.29%) among mothers and 0.56% (95%CI: 0.27%-0.85%) among children. The prevalence among children without any hepatitis B vaccination (HepB) was 4.62% (0.00%-9.72%). Among children with HBsAg-positive mother, prevalence was as high as 10.11% (3.85-16.38). Mother's HBsAg positivity and children never receiving any HepB were independently associated with HBsAg positivity of children. Interpretation: Having achieved the 2017 target of less than 1% prevalence among 5 year-old children, Cambodia should now focus on eliminating mother-to-child transmission of HBV. Moreover, the high prevalence among mothers suggests that routine screening with proper linkage to care and treatment is needed. Strengthening measures to improve vaccination coverage further and eliminate mother-to-child transmission by coordinated programming with other services offering additional HBV interventions will help move towards the global goal of HBV elimination by 2030. Funding Statement: This study was financially supported by 1) Hiroshima University, Department of Epidemiology, Infectious Disease Control and Prevention, Japan 2) Project Research Centre for epidemiology and prevention of viral hepatitis and hepatocellular carcinoma, Hiroshima University, Japan (Director: J. Tanaka) 3) World Health Organization Viral hepatitis Research Foundation of Japan (AAOHELP: Asia-Africa-Oceania Hepatitis Experts Linkage Promotion) 5) Institute of Immunology Co., Ltd., Japan 6) Ortho Clinical Diagnostics, Japan 7) PhoenixBio Japan Co., Ltd., Japan 8) Akane-kai Medical Corporation, Japan 9) Sysmex Corporation, Japan 10) Ohno-naika clinic, Japan 11) Sato Dental clinic, Japan Declaration of Interests: The authors declare no conflict of interests. Ethics Approval Statement: The study protocol was approved by the Cambodia National Ethics Committee for Human Research (392NECHR), the Ethics Review Committee for the WHO Western Pacific Region and the Ethical Committee for Epidemiology of Hiroshima University (E-573).