In this study, we investigated the seroprevalence of anti-hepatitis D virus (HDV) antibodies in hepatitis B surface antigen (HBsAg)-positive children after 25 years of obligatory vaccination of infants against hepatitis B virus. This cross-sectional study included 120 treatment-naïve HBsAg-positive children, with a male-to-female ratio of 1.8:1 and a mean age of 7.8 ± 3.8 years (range, 1-17 years). Mothers were positive for HBsAg in 96.6% of the cases. HBeAg-positive chronic infection was observed in 60% of the cases, HBeAg-positive chronic hepatitis in 12.5%, and HBeAg-negative chronic infection in 26.7%. Anti-HDV antibodies were not detected in any of the cases. Thus, there is a lack of anti-HDV antibodies in HBsAg-positive children, despite the current burden in adults.