Screening of blood reduces but does not eliminate the risk of hepatitis B virus (HBV) infection in multi-transfused thalassaemia patients. This study was done to evaluate efficacy of HBV vaccination on hepatitis B virus surface antigen (HBsAg) carrier rate in children with thalassaemia major receiving multiple blood transfusions. In a cohort study conducted at a hospital in Bahawalpur, Pakistan, during 2009–10, children with thalassaemia major aged < 60 months who received more than 24 blood transfusions and were HBsAg negative at the time of first blood transfusion were included. Of 196 unvaccinated children, the seropositive rate was 12.2%; while among 218 children vaccinated during the first year of life via the Pakistan Expanded Programme on Immunization, the seropositive rate was only 0.9%. The HBV vaccine was highly effective in reducing the HBsAg carrier rate in children with thalassaemia aged < 5 years.