OBJECTIVE: To assess the influence of body mass index (BMI) on fetal fraction increase with gestational age and on the rates of test failure. METHODS: We performed a cross-sectional study of consecutive singleton pregnancies in which cell-free DNA screening for fetal aneuploidies was performed from 10 weeks of gestation, between May 2013 and January 2018, at two fetal medicine clinics in Australia using one of two different platforms. Maternal characteristics, fetal fraction, and failure after a first attempt ("no-call") and after resampling ("test failure") were recorded. Body mass index was classified as normal (BMI less than 25.0), overweight (BMI 25.0-29.9), obesity class I (BMI 30.0-34.9), and obesity classes II and III (BMI 35.0 or greater). Fetal fraction and proportions of no-call results and test failure were compared between the groups. RESULTS: Of 14,223 singleton pregnancies included, 8,583 (60.3%) were screened with platform A and 5,640 (39.7%) with platform B. Median fetal fraction values were lower, and no-call and failure rates were higher in increased BMI groups across both platforms (P <.001 in both).When compared with women with normal BMIs, women with BMIs of 35 or greater had a significantly smaller increase in fetal fraction (0.1 vs 0.3 units/wk, P < .001, in both platforms). In this subgroup, when compared with women with normal BMIs, the odds ratios for a no-call result were 22.0 (95% CI 13.8-35.3, 16.6% vs 0.9%) and 8.0 (95% CI 4.1-15.6, 7.8% vs 1.0%) and for a failed test were 25.0 (95% CI 11.2-55.7, 6.4% vs 0.3%) and 5.8 (95% CI 2.0-17.3, 2.7% vs 0.5%) using platforms A and B, respectively. CONCLUSION: The increase in fetal fraction throughout gestation in women with BMIs of 35 or above is minimal. Postponing the test is unlikely to reduce test failure rates in this population.