Background. β-Thalassemia is a common inherited hemolytic disorder in Palestine. Red blood cell (RBC) transfusion is the principal treatment but it may cause RBC alloimmunization. This study was conducted to determine the prevalence and characteristics of RBC alloimmunization among thalassemic patients in northern governorates of Palestine. Methods. A prospective multicenter observational study was conducted in the thalassemia transfusion centers in the northern governorates of Palestine. The study included 215 thalassemia patients who received regular blood transfusions. Clinical and transfusion records of patients were examined. Antibody screening and identification was conducted using the microcolum gel technique. Results. Two hundred fifteen patients were included in the study. More than half (52.1%) of the patients were males. The median age of patients was 18 years (range: 12–24 years). The most frequent blood group was A (40.5%). Alloantibodies were detected in 12.6% of patients. Anti-D (33.3%), anti-K (25.9%) and anti-E (14.8%) were the most commonly isolated antibodies. There was no association between age, sex, starting age of transfusion, number of transfused units, history of splenectomy and alloimmunization. Conclusions. Anti-Rh and anti-K antibodies were common among this cohort of patients. Age, sex, starting age of transfusion, number of transfused units, and history of splenectomy could not predict the occurrence of alloimmunization.