OBJECTIVE The objective of the study was to evaluate the efficacy of maternal serum markers in detecting Down syndrome after 18 weeks of gestation in women who book late for maternity care in a large national retrospective study. STUDY DESIGN During the period 2007-2012, 27,648 women, regardless of maternal age (17.4% were 35 years old and over), were included in a late Down syndrome screening program (18(+0) to 35(+6) weeks) using the maternal serum markers alpha-fetoprotein and human chorionic gonadotrophin-beta. Samples were assayed in a single laboratory. A dataset of median markers previously established in our laboratory was used for risk calculation. The control group consisted of 27,648 women (14(+0) to 17(+6) weeks) randomly selected from the routine database. RESULTS When the later screening group was compared with the standard second-trimester control group, the median multiples of medians (1.01 vs 0.98 for alpha-fetoprotein, 1.03 vs 0.98 for human chorionic gonadotrophin-beta), median risks (1 of 2414 vs 1 of 2720), false-positive rates (11.1% vs 11.6%), and trisomy 21 detection rates (83.3% vs 85.7%) did not differ significantly. CONCLUSION Late Down syndrome maternal serum screening is feasible with a good sensitivity/specificity compromise throughout gestation and is of clinical value in late-booking women.