OBJECTIVES: The use of cell-free fetal deoxyribonucleic acid (DNA) requires the knowledge of variables that may influence the levels of cell-free DNA, such as maternal body mass index (BMI). MATERIAL AND METHODS: In this study, using 406 maternal blood samples from the second trimester of pregnancy, cell-free fetal DNA specific for the SRY and DYS14 loci and glyceraldehyde-3-phosphate dehydrogenase sequence were quantified by real-time polymerase chain reaction. RESULTS: No significant correlation was seen between the levels of cell-free fetal DNA and maternal BMI, whereas total cell-free DNA was significantly associated with maternal BMI at 20 to 21 weeks of gestation (P = .034) and at the end of pregnancy (R2 regression: 0.016, P = .014). CONCLUSION: Quantitative levels of cell-free fetal DNA are not affected by maternal BMI, whereas total DNA levels in the second trimester significantly correlate with maternal BMI at the moment of blood drawing and at delivery.