INTRODUCTION: HIV-1 and hepatitis B virus (HBV) share common routes of transmission and therefore co-infection is common. In 2019, an HIV-1 outbreak that resulted in >1000 children being infected, predominantly through nosocomial transmission, occurred in Sindh, Pakistan. We conducted a phylogenetic and drug resistance analysis of the HBV Reverse Transcriptase (RT) gene in children with HIV-1 and HBV co-infection. METHODOLOGY: Blood samples were collected from 321 children with HIV who were recruited as part of a study to investigate the HIV-1 outbreak. All samples were tested for HBV surface antigen (HBsAg) using an ELISA assay, and positive samples were used to amplify and sequence the HBV RT gene. The phylogenetic relationship between sequences was analyzed, and drug- and vaccine- resistance mutations in the RT gene were explored. RESULTS: Of 321 samples, 23% (n = 75) were positive for HBsAg on ELISA. Phylogenetic analysis of the sequences revealed that 63.5% of HBV sequences were sub-genotype D1, while the rest were sub-genotype D2. Cluster analysis revealed grouping of sub-genotype D1 sequences exclusively with Pakistani sequences, while clustering of sub-genotypes D2 predominantly with global sequences. The 236Y mutation associated with resistance to tenofovir was observed in 2.8% of HBV sequences. Additionally, seven vaccine escape mutations were observed, the most common being 128 V. CONCLUSION: Our study suggests ongoing transmission of HBV D1 and D2 sub-genotypes in the HIV-1 co-infected population, likely nosocomially, given common routes of HVB and HIV-1 transmission. The prevalence of major HBV drug- and vaccine-resistant mutations remains low. Surveillance for further transmissions and the possible emergence of major drug- or vaccine-resistant variants is required.