BACKGROUND: ABO subgroups are obstacles in blood typing and cross matching in the process of pre-transfusion. Correct ABO typing is important for ensuring safety of a transfusion. The ABO subgroups contain a lesser amount of antigen on red cells and this is a common cause of discrepancy between the results of cell and serum tests. This study was performed to analyze the frequency and distribution of the ABO subgroups in Korean blood donors. METHODS: ABO blood grouping was performed by an automated analyzer (PK7200/7300, Olympus, Japan) for the 3,397,983 donors from January 2007 to December 2009. The 1,868 donor samples that were suspected to have ABO subgroups were tested with adsorption/elution techniques. The results of the tests were analyzed and we received approval of the Institutional Review Board of the Korean Red Cross to publish this study. RESULTS: 1,771 donors out of the 1,868 donors who underwent the tests were finally identified as having ABO subgroups. The kinds of identified ABO subgroups were as follows; 118 donors with the A subgroup (A2, A3, Ax, Am, Ael), 237 donors with the B subgroup (B3, Bx, Bm, Bel) and 1,416 donors with the AB subgroup (A2B, A2B3, A1B3, AmB, A1Bm, A1Bel, AxB, A1Bx). The most frequently observed subgroups were A2B (496/1,868, 26.55%) and then A2B3 (457/1,868, 24.46%). The subgroups of 97 donors (5.19%) were not identified. CONCLUSION: This study was meaningful to provide data on the frequency and distribution of the ABO subgroups of Korean blood donors. A more sensitive technique such as genotyping could be useful to resolve cases with an unidentified subgroup.