HIV/HBV co-infection is highly prevalent in sub-Saharan Africa. The aim of this study was to determine if the use of triple combination lamivudine-containing prophylaxis for the prevention of mother-to-infant HIV transmission was associated with the emergence of lamivudine HBV mutations. The study included 21 pregnant co-infected women in Malawi who received either zidovudine or stavudine plus lamivudine and nevirapine from week 25 of gestation until 6 months after delivery or indefinitely if they met the criteria for treatment (CD4+ <350/mm3). HBV-DNA was determined using the Roche COBAS assay. Resistance mutations were assessed by the Trugene assay (Siemens Diagnostics). At baseline 33% of the women were HBeAg positive and had HBV-DNA > 104 IU/ml. Median CD4 count was 237 cells/mm3 and median HIV-RNA was 3.8 log10 copies/ml. After a median of 259 days of treatment, HBV-DNA was detectable in 9 out of 21 patients (42.8%). In three cases the HBV-DNA level was >104 IU/ml. Resistance mutations (M204I in five cases and L180M + M204I/V in one case) were present in 6 (28.6%) patients. Women with a resistant virus had significantly higher baseline HBV-DNA levels than those not developing resistance (1.1 × 107 IU/ml vs. 20.8 IU/ml, P = 0.022). Levels of ALT and AST were higher in women with resistant viruses compared to those retaining a wild-type virus. A high rate of lamivudine resistance was seen in this cohort of pregnant women. Follow-up of these patients will clarify if the presence of resistance has a significant impact on liver disease. J. Med. Virol. 84:1553–1557, 2012. © 2012 Wiley Periodicals, Inc.