The use of current screening for Down syndrome is relatively effective and can detect up to 90% of cases with a false-positive rate of about 5%. However, follow-up invasive procedures are associated fetal loss in as many as 1 in 200 tests. Alternative noninvasive diagnostic tests have been investigated that can detect fetal cell-free DNA in maternal blood. In 2008, 2 reports described the use of such a test, massively parallel shotgun sequencing (MPSS), which is an internally validated test based on next-generation sequencing. Subsequent studies with the MPSS test were promising and suggested a detection rate of at least 98% at a false-positive rate of 2% or lower. However, sample sizes were small (13–86 Down syndrome cases), and the test was not performed in Clinical Laboratory Improvement Amendments (CLIA)-certified laboratories. This blinded, nested case-control study analyzed the use of MPSS within a cohort of 4664 pregnancies. Women at high risk for Down syndrome were enrolled at 27 prenatal diagnostic centers worldwide. The test was evaluated in 212 Down syndrome and 1484 matched euploid pregnancies. Similar numbers of Down syndrome cases were diagnosed in the first and second trimester. None of the samples had been previously tested. A CLIA-certified commercial laboratory performed the primary testing. A subset of samples was sent to a CLIA-certified university laboratory for confirmatory testing. MPSS testing correctly identified 209 of 212 Down syndrome fetuses; the detection rate was 98.6%, with a 95% confidence interval of 95.9–99.7. The false-positive rate was 0.2% (3/1471), with a 95% confidence interval of <0.1–0.6. Testing failed in 13 euploid pregnancies (0.8%). After testing was concluded but before unblinding, the primary testing laboratory adjusted the chromosome 21 data for guanine cytosine base content to assess alternative methods of interpreting the MPSS results. Adjusting chromosome 21 counts for guanine cytosine base content had the largest impact on improving test performance (reduced false-negative and false-positive results). These findings show that measuring maternal plasma DNA in a large cohort of high-risk pregnancies can identify nearly all cases of Down syndrome at a very low false-positive rate. Although confirmation by invasive testing is needed, data from this and previous studies suggest the benefit of this test in women at high risk of Down syndrome.