Hepatitis B virus (HBV) and human immunodeficiency virus (HIV) are hyperendemic in sub-Saharan Africa. The HBV genotypes prevailing in HIV-infected Africans are unknown. Our aim was to determine the HBV genotypes in HIV-infected participants and to identify clinically significant HBV mutations. From 71 HBV DNA(+ve) HIV-infected participants, 49 basic core promoter/precore (BCP/PC) and 29 complete S regions were successfully sequenced. Following phylogenetic analysis of 29 specimens in the complete S region, 28 belonged to subgenotype A1 and one to D3. Mutations affecting HBeAg expression at the transcriptional (1762T1764A), translational (Kozak 1809-1812, initiation 1814-1816, G1896A with C1858T), or post translational levels (G1862T), were responsible for the high HBeAg-negativity observed. The G1862T mutation occurred only in subgenotype A1 isolates, which were found in one third (7/21) of HBsAg(-ve) participants, but in none of the 18 HBsAg(+ve) participants (p<0.05). Pre-S deletion mutants were detected in four HBsAg(+ve) and one HBsAg(-ve) participant/s. The following mutations occurred significantly more frequently in HBV isolated in this study than in strains of the same cluster of the phylogenetic tree: ps1F25L, ps1V88L/A; ps2Q10R, ps2 R48K/T, ps2A53V and sQ129R/H, sQ164A/V/G/D, sV168A and sS174N (p<0.05). ps1I48V/T occurred more frequently in females than males (p<0.05). Isolates with sV168A occurred more frequently in participants with viral loads >200 IU per ml (p<0.05) and only sS174N occurred more frequently in HBsAg(-ve) than in HBsAg(+ve) individuals (p<0.05). Prior to initiation of ART, ten percent, 3 of 29 isolates sequenced, had drug resistance mutations rtV173L, rtL180M+rtM204V and rtV214A, respectively. This study has provided important information on the molecular characteristics of HBV in HIV-infected southern Africans prior to ART initiation, which has important clinical relevance in the management of HBV/HIV co-infection in our unique setting.