Traditional blood group ABO serology is based on immunoreactivity with the carbohydrate determinants A, B and H antigens. Recent advances at the DNA level of the ABO genes have provided a molecular genetic model for the ABO polymorphism. This genetic model has to date only been tested on a limited basis. The present study was initiated to evaluate the universality of the proposed genetic model on a larger group of serologically defined ABO phenotypes. Three hundred healthy Danish blood donors were analysed (A: 50, B: 50, AB: 50, O: 150) by PCR amplification followed by diagnostic restriction enzyme cutting. In all cases A, B, and AB at least one allele of correctly predicted status was found. However, in O phenotype individuals, 11 out of 150 carried one allele discordant to the proposed genetic model. This novel O allele (3.7% allele frequency) was further characterized by diagnostic restriction enzyme analysis in two positions divergent between A and B alleles and by DNA sequencing of the two major exons. The novel O allele is termed O² as it typed as B in nucleotide position 526 and as A in positions 703, 796, and 803, in contrast to the most predominant O allele termed O¹, which types as A in all 4 positions. The structural defect in the O² allele appears to be an additional substitution at nucleotide position 802. The results clearly demonstrate that with the addition of the two distinctly different O alleles, O¹, O², the previously proposed molecular genetic basis of the ABO polymorphism is quite valid. More importantly the determined characteristics of these two O alleles have practical implications in ABO genotyping, because it establishes within the limits of the number of samples tested that ABO genotypes can be assessed directly by non‐allele specific PCR amplification and restriction enzyme analysis.