OBJECTIVE: To estimate whether multiplex ligation-dependent probe amplification (MLPA), a molecular technique used for detecting the most common chromosomal aneuploidies, is comparable with karyotyping for the detection of aneuploidies of chromosomes X, Y, 13, 18, and 21 in routine clinical practice and to estimate the costs differences of both techniques. METHODS: In this prospective, nationwide cohort study, we consecutively included 4,585 women who had an amniocentesis because of their age (36 years or older), increased risk after prenatal screening, or maternal anxiety. Amniotic fluid samples were tested independently with both MLPA and karyotyping. The primary outcome was diagnostic accuracy of MLPA to detect aneuploidies of chromosomes X, Y, 13, 18, and 21. Secondary outcome Measures were turnaround time for test results and costs. A sample size was calculated using a critical noninferiority margin of 0.002; therefore, at least 4,497 paired test results were needed (one-sided alpha 0.05, power 0.90). RESULTS: Diagnostic accuracy of MLPA was 1.0 (95% confidence interval [CI] 0.99-1.0), sensitivity was 100% (95% CI 0.96-1.0) and specificity was 100% (95% CI 0.999-1.0). Diagnostic accuracy of MLPA was statistically similar (noninferior) to that of karyotyping (P<.001). In 75 cases, MLPA failed (1.6%); karyotyping failed once (0.02%). Compared with karyotyping, MLPA shortened the waiting time by 14.5 days (P<.001, 95% Cl 14.3-14.6) and cost less (-47, P<.001). CONCLUSION: In routine clinical practice, diagnostic accuracy of MLPA for detection of trisomies X, Y, 13, 18, and 21 is comparable with that of karyotyping, and it reduces waiting time at lower costs. (Obstet Gynecol 2010,115:297-303)