Background : Antibody screening and identification tests are indispensable test to protect hemolytic transfusion reaction . Due to increase of transfusion and development of new techniques for detection of antibodies, the incidence of unexpected antibodies may tend to increase compared with the past. Methods : We evaluated the frequency and distribution of unexpected antibodies by two methods from 53,167 sera. The tube(saline, albumin, and antiglobulin phase) methods were subjected to the sera from Jan 1998 to April 2000, and gel agglutination methods with DiaMed-ID system (DiaMed, Murten, Switzerland) were subjected to the sera from May 2000 to Feb 2004. Results : Out of all 53,167 serum samples, positive results were obtained from 177 sera(0.33%). Antibody that was detected most frequently was anti-E(46 sera, 25.9%), followed by anti-(43 sera, 22.6%), anti- and anti-c. In the tube method, 55 cases(83.3%) were detected at antiglobulin phase. Among the patients with unexpected antibodies, 85 cases(54.5%) had the history of previous transfusion. Conclusion : Unexpected antibodies that were detected in this study were mostly significant antibodies in clinical. There was no difference of the frequency according to the method. Unexpected antibodies were associated with multiple transfusion. Therefore antibody screening and identification tests are critical step in pre-transfusion tests.