Maternal plasma human immunodeficiency virus (HIV) type 1 RNA load has a role in perinatal transmission, but significant overlap in the range of plasma virus loads among transmitters and nontransmitters is often observed, which makes it difficult to predict transmission outcome. We measured several virus markers in a drug-naive population of HIV-1-infected mothers in Botswana. Maternal plasma HIV-1 RNA load, peripheral blood mononuclear cell-associated blood HIV-1 DNA load, and cervicovaginal fluid (CVF) HIV-1 DNA load were determined using quantitative real-time polymerase chain reaction. The overall rate of transmission among these mother-infant pairs was 35.7%. Median infant age was 2.5 months. An association between increased plasma HIV-1 RNA load and perinatal transmission was observed (odds ratio [OR], 2.20/1-log virus load; 95% confidence interval [CI], 1.15-4.18). However, the association between increased blood HIV-1 DNA load and perinatal transmission was stronger (OR, 10.30; 95% CI, 2.11-50.38). When blood HIV-1 DNA load was combined with CVF HIV-1 DNA load, the association with transmission increased (OR, 25.0; 95% CI 2.73-228.60).