BackgroundIdentifying genetic variants of the ABO gene may reveal new biologic mechanisms underlying variant phenotypes of the ABO blood group. We report the molecular genetic analysis of 322 apparently unrelated ABO subgroup individuals in an estimated 2.1 million donors.Study Design and MethodsWe performed phenotype investigations by serology studies, analyzed the DNA sequence of the ABO gene by direct sequencing or sequencing after cloning, and evaluated promoter activity by reporter assays.ResultsIn 62 rare ABO alleles, we identified 29 novel ABO subgroup alleles in 43 apparently unrelated subgroup individuals and their four available pedigrees. Of these alleles, one was a deletion‐mutation allele, four were hybrid alleles, and 24 were point‐mutation alleles. Most of the point mutations were detected in Exons 6 to 7, while several others were also detected in Exons 1 to 5 or splicing regions. One ABO promoter mutation, −35 to −18 del, was found and verified to reduce promoter activity, as determined by dual luciferase assays. Two mutations, 7G>T and 52C>T, carrying the premature terminal codons E3X and R18X in the 5′‐region, were found to be associated with the very weak ABO subgroups “Ael” and “Bel.”ConclusionTwenty‐nine ABO subgroup alleles were newly linked to different kinds of ABO variations. We provide the first evidence that promoter abnormality is involved in the formation of weak ABO phenotypes. We also described the first naturally occurring ABO alleles with premature terminal codons in the 5′‐region that led to Ael and Bel phenotypes.