The phenotypic distributions of observed numbers of ABO blood groups in a Kuwaiti sample population of 18,558 subjects are 4962 (26.7%) with A, 4,462 (24.1%) with B, 858 (4.6%) with AB, and 8,276 (44.6%) with 0. The calculated gene frequencies are 0.6678 for ABO*O, 0.1768 for ABO*A, and 0.1554 for ABO*B. Molecular genotyping of the ABO blood group system in a Kuwaiti sample population was determined using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. The positions of nucleotides 258 and 700 of cDNA from A transferase were amplified by PCR. The amplified DNA was subjected to RFLP analysis to distinguish A, B, and O alleles. Blood samples of known ABO phenotype from 101 healthy unrelated Kuwaiti individuals (A, 29; B, 23; AB, 14; O,35) were used. Two DNA fragments of the ABO locus were designed to be amplified by 2 pairs of primers. To identify the 258th nucleotide, a 199- or 200-bp DNA fragment was amplified by PCR and digested with KpnI. For the 700th nucleotide, a 128-bp DNA fragment was amplified by PCR and digested with AluI. By analyzing the electrophoresis patterns,ABO genotypes were conclusively determined by examining the DNA fragments. The ABO genotypes of the known 101 samples were as follows: AA, 4.30%; AO, 24.41%; BB, 4.16%; BO, 24.2%; AB, 8.46%; and 00, 34.65%. These results were confirmed statistically using the calculated frequencies of IA, IB, and IO alleles.