Leucodepletion has been shown to reduce human leucocyte antigen immunization, but studies on the effect of leucodepletion on red cell alloimmunization reported discordant results. We conducted a retrospective multicentre study to determine whether prestorage filter leucodepletion alters the development of clinically significant red blood cell alloimmunization against the Rhesus, Kell, Duffy, Kidd and MSs blood group systems. Two periods were investigated, 2 years before and 2 years after universal leucodepletion. Comparisons were made between the transfused patient cohorts. To control for changes not related to leucoreduction, we compared antibody incidence with antibody prevalence in the two study periods. Newly detected antibodies (n = 4770) were found in 4115 patients from 19 participating hospitals. Of these, 857 antibodies in 659 patients were because of transfusions given in the study periods. The immunization risk was 0.13% for both periods. No differences were found regarding incidence of new antibodies, nor for patients regarding age, sex, previous antibodies, multiple antibodies, additional antibodies, number of transfusions, transfusions episodes and days from transfusion to date of immunization. In conclusion, compared with buffy-coat leucoreduction, universal prestorage filter leucodepletion did not alter the development of clinically significant red blood cell alloimmunization.