Abstract: To determine the phenotypic, allelic frequencies and the genotypes of ABO blood groups in a Jordanian population, samples of 12215 randomly healthy Jordanian voluntary blood donors during the period 1998-2003 were taken from the National Blood Bank donor registry, Amman, Jordan. The results of the phenotypic distribution indicated that 4686 (38.36%) of the donors were type A, 4473 (36.62%) O, 2203 (18.04%) B and 853 (6.98%) AB. The gene frequencies were 0.6052 for Io allele, 0.2607 for Ia allele and 0.1341 for Ib allele. Using PCR-RFLP technique, two separate segments of the transferase gene containing nucleotide 261 in exon 6 and nucleotide 703 in exon 7 of the ABO gene locus were amplified and their products were analyzed with two restriction enzymes (KpnI and AluI). The electrophoresis patterns of 105 samples showed that ABO genotypes were AA: 6 (5.714%), AO: 35 (33.333%), BB: 1 (0.953%), BO: 14 (13.333%), AB: 10 (9.524%) and OO: 39 (37.143%).