Malaria infection during pregnancy is associated with adverse consequences including low birth weight (LBW) and maternal anemia, particularly in primigravidae and secundigravidae. In preparation for a clinical trial of the efficacy of chloroquine (CQ) and sulfadoxine-pyrimethamine (SP) containing prevention regimens during pregnancy, we conducted a one-year cross sectional study in Koro and Bandiagara, Mali using an assessment methodology developed by the Centers for Disease Control and Prevention (CDC) to generate basic data on malarial burden during pregnancy. Two hundred and sixty-one and 192 women were enrolled in Koro and Bandiagara, respectively. Rates of placental parasitemia were 17.1 and 42.3% in Koro and Bandiagara, respectively, despite high (70–80%) use of preventive medication (mainly CQ). Low gravidity (1st and 2nd pregnancies) was associated with peripheral (p < 0.001) and placental (p < 0.001) malaria only in Bandiagara, whereas it was associated with low birth weight in both sites (p < 0.001 in Koro and p = 0.002 in Bandiagara). First and second pregnancies were the most important characteristics associated with placental malaria (RR = 2.78, 95%CI 1.81–4.29) and (ARR = 2.06, 95%CI 1.03–4.15) and low birth weight (RR = 4.26, 95%CI 2.50–7.27) and (ARR = 4.51, 95%CI 2.55–8.00). Birth during the rainy season was associated with placental infection in univariate analysis. Characteristics such as younger age, having fever during pregnancy, and unmarried status were associated with low birth weight only in univariate analysis and singleton premature delivery and low gravidity were associated with low birth weight in both univariate and multivariate analysis. Data from this assessment demonstrated the high burden of malaria during pregnancy in Mali. Results had been used by researchers as local reference data and by ministry of health for to stop recommending CQ prophylaxis. The methodology could be used by other malaria-endemic countries to direct their national malaria program efforts.