Background: An exact ABO blood type is essential for transfusion therapy. Genetic structures of ABO blood group and subgroup have been investigated and about 100 ABO alleles have been reported worldwide. This study was performed to investigate the molecular characteristics of A subgroups in the Korean population. Methods: Nine A and five AB subgroups were collected from patients and from blood donors of Korean Red Cross blood centers after serological ABO blood group typing. On these samples, allele-specific polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), direct sequencing of exon 6 and 7, and allele separation were performed for ABO gene analysis. Results: The ABO PCR-RFLP genotyping results of 13 samples among the provisional 14 A subgroups were identical with their phenotypes. ABO*A205 allele was observed in an Aint subgroup. Two new A alleles that showed 784G>A base change and 990C>T of intron 6. And a polymorphism of 532C>T in A(pro) intron 5 were also discovered. Conclusion: Through the molecular analysis of this study, serologically unidentified A subgroups were obviously identified. And the new alleles only observed in the Korean A subgroups were recognized.