While many factors contribute to mother-to-child transmission (MTCT) of HIV-1, maternal plasma HIV-1 RNA viral load (RNA-VL) has been consistently found as the main risk factor, including when antiretroviral prophylaxis was used to prevent MTCT. However the predictive value of RNA-VL is poor. A recent study of HIV-1-positive pregnant women who did not receive antiretroviral prophylaxis reported an association between HIV-1 DNA viral load (DNA-VL) and MTCT that was stronger than the association between RNA-VL and MTCT. We sought to determine if HIV-1 DNA-VL was independently associated with MTCT of HIV in a population of women who received zidovudine prophylaxis during pregnancy and whose infants received zidovudine after birth. Patients were 33 non-breastfeeding transmitting (TR) and 33 nontransmitting mothers (NTR) from Perinatal HIV Prevention Trial (PHPT-1), a multicenter clinical trial conducted in Thailand comparing zidovudine prophylaxis durations to prevent MTCT. TR and NTR mothers were matched according to baseline RNA-VL. Maternal peripheral blood mononuclear cell (PBMC)-associated HIV-1 DNA was extracted from whole blood, and DNA-VL was established by quantitative real-time polymerase chain reaction. We found that TR had a significantly higher cell-associated HIV-1 DNA viral load than did NTR. Median TR DNA-VL was 2.54 log(10) copies per microgram PBMC DNA, while it was 2.28 log(10) copies per microgram PBMC DNA in NTR (Wilcoxon p = 0.02). In summary, HIV-1 DNA viral load was associated with MTCT in a population of women who received antiretroviral prophylaxis during pregnancy, independently from RNA viral load.