Hepatitis B virus genetic diversity (HBV) evaluation is scarcely done in Tanzania, imposing a crucial knowledge gap toward elimination of HBV infection by 2030. This cross-sectional study was conducted on purposively selected 21 plasma samples with high HBV-deoxyribonucleic acid (DNA) levels of >
300,000IU/mL. DNA extraction was done using Qiagen DNA Blood Mini Kit (Qiagen, Hilden, Germany). Partial amplification of 423 bp of pol gene, sequencing and analysis
and statistical analysis by STATA version 15 were done. These patients had mean age of 41 ± 11 years with HBV-DNA median of 979 [185.5-8457.5] IU/mL. The genotypes detected were HBV/A
76.2% (16/21), HBV/D
19% (4/21), and lastly HBV/G
4.8% (1/21). Most of the HBV/As and all of the HBV/Ds identified in this study did not cluster with HBV/As and HBV/Ds from other parts of the world. Overall, 19% (4/21) of the patients had HBV escape mutations (T123V, Y134N, P120T and T123A). In conclusion, HBV/A and HBV/D are predominant over time in North-western Tanzania. Most HBV/A and all HBV/D are unique to Tanzania as had been previously reported. However, the pattern of hepatitis B virus genetic diversity is changing in Northwestern Tanzania with occurrence of HBV/G as new genotype in the region.