Hepatitis B virus (HBV) infection is one of the major public health problems in Malaysia. It remains the most common permanent deferral among blood donors. In Malaysia, the national vaccination programme has been introduced since 1989 to prevent HBV transmission. The objective of this study is to determine the prevalence and associated risk factors of HBV infection among first-time blood donors after the implementation of the national hepatitis B vaccination programme. Methods: This is a retrospective cohort study involving tracing of the database of National Blood Centre Malaysia. The record of first-time blood donors who had donated between 1st January 2010 and 31st December 2015 and were screened HBV positive was reviewed and analysed. Results: There were 376,737 first-time donors who had donated blood and 575 of them screened positive for HBV. The overall prevalence of seropositive for hepatitis B was 0.15%. The prevalence was higher at 0.23% among donors born before the year 1989 (pre-vaccination era) compared to 0.05% among donors born in and after the year 1989 (post-vaccination era). Perinatal transmission was found to have 15 times higher odds of developing HBV infection as compared to those who had the combination of risk factors among those born after the year 1989 (adjusted OR=14.95, 95% CI 1.80=124.01). Conclusion: The implementation of the national vaccination programme reduced the prevalence of hepatitis B among donors who received vaccination at birth compared to those who did not.