The study evaluated and compared the prevalence of anti-HBs and exposure to hepatitis B virus (HBV) in vaccinated South African babies aged between 5 and 24 months from the Expanded Programme on Immunisation clinic [EPI group] and paediatric outpatient clinic [OPD group], and results were stratified by HIV status. A total of 303 (243 EPI group and 60 OPD group) babies were studied. All sera were tested for anti-HBs, HBsAg and anti-HBc, while IgM anti-HBc and HBV DNA were only tested in samples positive for HBsAg and/or anti-HBc. Overall, there was a gross difference in the prevalence of anti-HBs marker between the EPI and OPD groups. The EPI group demonstrated higher levels of seroconversion (89.3% vs. 81.7%; p=0.105) and seroprotection rates (86.0% vs. 75.0%; p=0.038), compared to the OPD babies. When the overall results were stratified by HIV status, seroprotection was 85.7% for the HIV-negatives and 78.1% for the HIV-positives, although this was not statistically significant (p=0.125). The seroprotection rates were almost comparable between the HIV-positives (84.3%; n=51) and the HIV-negatives (86.5%; n=192) (p=0.695) in the EPI group. In contrast, reduced seroprotection rates were observed between the HIV-positives (63.6%; n=22) and HIV-negatives (81.6%; n=38) in the OPD group, although this was not statistically significant (p=0.123). Interestingly, no HBsAg or anti-HBc marker was detected in the OPD group, compared to total exposure rate of 4.9% (HBsAg carriage was 1.2%) in the EPI group.