ObjectiveTo evaluate the effect of red blood cell (RBC) antibody screening in the 27th week of pregnancy in Rhc‐negative women, on detection of alloimmunisation, undetected at first trimester screening (‘late’ alloimmunisation), and subsequent haemolytic disease of the fetus and newborn (HDFN), to assess risk factors for late alloimmunisation.DesignProspective cohort and nested case–control study.SettingThe Netherlands.PopulationTwo‐year nationwide cohort.MethodsProspective inclusion of Rhc‐negative women with negative first trimester screening and of screen‐negative controls. Assessment of incidence and numbers needed to screen (NNS) of late alloimmunisation and HDFN; logistic regression analysis to establish risk factors for late alloimmunisation.Main outcome measuresLate alloimmunisation, HDFN.ResultsLate alloimmunisation occurred in 99 of 62 096 (0.159%) Rhc‐negative women; 90% had c/E antibodies and 10% non‐Rhesus antibodies. Severe HDFN (fetal/neonatal transfusion) occurred in two of 62 096 (0.003%) of Rhc‐negative women and 2% of late alloimmunisations; moderate HDFN (phototherapy) occurred in 20 children [22.5%; 95% confidence interval (CI), 13.8–31.1%]. Perinatal survival was 100%. The NNS to detect one HDFN case was 2823 (31 048 for severe, 3105 for moderate HDFN). Significant risk factors were former blood transfusion [odds ratio (OR), 10.4; 95% CI, 1.14–94.9], parity (P−1: OR, 11.8; 95% CI, 3.00–46.5; P > 1: OR, 7.77; 95% CI, 1.70–35.4) and amniocentesis/chorionic villus sampling during current pregnancy (OR, 9.20; 95% CI, 1.16–72.9).ConclusionsAdditional screening of Rhc‐negative women improved the detection of late alloimmunisation and HDFN, facilitating timely treatment, with a NNS of 2823. Independent risk factors for late alloimmunisation were blood transfusion, parity and chorionic villus sampling/amniocentesis in the current pregnancy. The occurrence of most factors before the current pregnancy suggests a secondary immune response explaining most late alloimmunisations.Tweetable abstractThird trimester screening for alloimmunisation in Rhc‐neg women improves detection and treatment of severe HDFN.