Objective: To evaluate the clinical validity of a new ultrahigh-throughput non-invasive prenatal test (NIPT) based on combinatorial probe–anchor ligation (cPAL) sequencing of cell-free fetal DNA (cffDNA) using centralized testing. Methods: Maternal plasma samples were obtained from 10 594 singleton pregnancies in high-risk populations at 20 centers in China, including 8155 that were collected retrospectively and 2439 prospectively. Fetal outcome data and karyotyping results were documented as gold standard and were double blinded during NIPT. The clinical performance of the ultrahigh-throughput sequencing method, cPAL, for NIPT was validated by evaluating its sensitivity, specificity and positive predictive value (PPV) in detecting trisomies 21, 18 and 13 as the centralized testing mode in the reference laboratory. To ensure stable and reproducible performance of centralized cPAL-based NIPT in detecting trisomies, a series of quality-control systems, including sequencing of two sets of artificial samples, were employed and evaluated. Results: Ten prospective cases were excluded from the study because of incomplete clinical data. Four prospective samples failed to generate a NIPT result due to assay failure, presenting a failure rate of 0.16% (4/2429). A total of 168 retrospective cases and 47 prospective cases had a positive NIPT result for trisomy, giving respective positive rates of 2.06% and 1.94%. Four false-positive and no false-positive cases were observed in the retrospective and prospective groups, respectively, resulting in PPV of 97.62% (95% CI, 94.02–99.35%) and 100% (95% CI, 92.45–100%), respectively. In the retrospective group, sensitivity and specificity were, respectively, 100% (95% CI, 97.07–100%) and 99.98% (95% CI, 99.94–100%) for trisomy 21, 100% (95% CI, 97.75–100%) and 99.98% (95% CI, 99.94–100%) for trisomy 18, and 100% (95% CI, 15.81–100%) and 100% (95% CI, 99.95–100%) for trisomy 13. In the prospective group, sensitivity and specificity were, respectively, 100% (95% CI, 90.75–100%) and 100% (95% CI, 99.85–100%) for trisomy 21, 100% (95% CI, 63.06–100%) and 100% (95% CI, 99.85–100%) for trisomy 18, and 100% (95% CI, 2.50–100%) and 100% (95% CI, 99.85–100%) for trisomy 13. Conclusion: In this multicenter study with a full quality-control system, NIPT by centralized cPAL-based testing showed high stability and performance comparable to those of previous validation studies in high-risk populations. Copyright © 2016 ISUOG. Published by John Wiley & Sons Ltd. Copyright © 2016 ISUOG. Published by John Wiley & Sons Ltd.