Background <p>Anti-D immunoglobulin administered in the early third trimester to Rhesus D negative women with Rhesus D incompatibility is widely assumed to provide protection against silent fetomaternal haemorrhage until delivery. However, previous studies in singleton pregnancies demonstrated that passively acquired anti-D is frequently no longer detectable by the time of delivery. The aim of this study was to assess whether the proportion of women with detectable passively administered anti-D antibodies in maternal serum at delivery differs between twin and singleton pregnancies.</p> Materials and methods <p>In this retrospective cohort study, twin and singleton pregnancies with Rhesus incompatibility that delivered at Kayseri City Hospital, Obstetrics and Gynecology Clinic between January 2020 and July 2025, in women who had not previously undergone alloimmunization and received anti-D immunoglobulin prophylaxis at 28 weeks of gestation, were compared in terms of antibody persistence determined by the indirect Coombs test of maternal serum at delivery.</p> Results <p>The indirect Coombs test performed at delivery was found to be negative in 50 (70.4%) of 71 twin pregnancies and in 100 (56.5%) of 177 singleton pregnancies. This difference was found to be statistically significant.</p> Conclusions <p>Twin gestations showed a lower frequency of detectable passive anti-D at delivery compared with singletons. This represents a pharmacokinetic/surrogate laboratory finding and should not be interpreted as evidence of reduced clinical effectiveness of prophylaxis. Prospective studies with serial measurements and clinical outcomes are warranted.</p>