Introduction: This study aimed to determine the seroprevalence of hepatitis B virus (HBV) infection in children and females in the middle Black Sea Region of Turkey and to assess the impact of the universal infant hepatitis B vaccination program started in 1998. Methodology: The laboratory records of 12,057 patients who attended the Samsun Maternity and Children’s Hospital between January 2007 and November 2009 were evaluated retrospectively. In this period, hepatitis B surface antigen (HBsAg), the antibody to hepatitis B surface antigen (anti-HBs), was studied from serum samples using the enzyme linked immunosorbent assay method. Results: In the total population, HBsAg seropositivity was found to be 3.8% (456/12010); anti-HBs was 32.6% (3526/10800). HBsAg was 3.5%, in the 0-14 year age group, 8.2% in the 15-18 group, 3.3% in the 19-49 group, and 8.0% in the over 49 group. The seropositivity of HBsAg decreased by half in patients between zero and 14 years of age (4.2%) who were included in the universal vaccination program when compared with those in the 15-18 year group (8.2%) who were not included in the program. There was a significant difference in HBsAg seropositivity between boys (5.8%) and girls (3.9%) (p < 0.05). Conclusions: According to the hepatitis B seroprevalence records of our region, HBsAg was 3.5% in the 0-14 year age group, 8.2% in the 15-18 year group. It may therefore be suggested that, since 1998, the vaccination program in our region has been successful.