Molecular genotyping of the ABO alleles has been widely used in ABO subgroups analysis and has been able to solve the rare ABO blood grouping discrepancies. The genotypes of sixty-one A2B phenotype donors recruited from the middle and south of Taiwan were analyzed by means of molecular methods. The A2B phenotype was initially identified by serological test. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used to screen the ABO alleles at nucleotides (nt) 261 and 703 based on the nt differences found in the ABO alleles. The subgroups of the A2 allele were determined by the PCR-RFLP and direct sequencing methods. The discrepancies between the phenotype and genotype of the A2B were then studied by subcloning and nucleotide sequence analysis. Our results show that 55 of the 61 A2B donors (90%) are A205/B allele and two are A201/B allele. Four cases were heterozygotes of the cis-AB/O or B alleles. Two were cis-AB04/O allele, one was cis-AB01/O allele and the other was cis-AB02/B allele. In conclusion, most A2B genotypes belong to the A205/B allele in Taiwan. In this study, we report for the first time the presence of the A205, A201, and cis-AB02 alleles in Taiwan.