In Thailand and adjacent countries, most of the β-thalassemia genes are β0-thalassemia mutations that prevent the production of Hb A. We propose the quantitation of the Hb A fraction in fetal blood in the mid-trimester of pregnancy by automated high performance liquid chromatography as a reasonable prenatal diagnostic method to be applied in areas with limited laboratory facilities. Forty pregnant women at risk of delivering a child with β-thalassemia major were identified using an erythrocyte osmotic fragility test and quantitation of Hb A2. Cordocentesis was performed at the gestational age of 18-22 weeks and fetal blood was analyzed for hemoglobin fractions by automated high performance liquid chromatography. The β-globin gene mutations were characterized by β-globin gene sequencing. The 4 bp deletion at codons 41/42 (-TTCT) was the most frequent of the 40 β-thalassemia mutations observed (20/40=50%), followed by the splice site mutation IVS-I-1 (G→T) (7/40=17.5%), the nonsense mutation at codon 17 (A→T) (7/40=17.5%), the nonsense mutation at codon 35 (C→A) (3/40 = 7.5%), and the β+-thalassemia promoter mutation at -28 (A→G) (3/40=7.5%). High performance liquid chromatography revealed nine fetuses which had only Hb F and no Hb A. All were homozygotes or compound heterozygotes for β0-thalassemia mutations. In the remaining 31 fetuses, a Hb A peak was present in the chromatograms. One fetus with 0.5% Hb A was a compound heterozygote for the -28 (A→G) and codons 41/42 (-TTCT) mutations. In the remaining 30 fetuses, the Hb A values ranged between 0.8 and 7.4%. Twenty of these, with a Hb A concentration of 1.824±0.49% (range 0.8-2.8%), were β-thalassemia heterozygotes. The remaining 10 fetuses had Hb A values of 4.894±1.47% (range 2.9-7.4%) and normal β-globin genes. The absence of Hb A in homozygotes or compound heterozygotes for β0-thalassemia mutations and the presence of measurable amounts of Hb A in heterozygotes and normal homozygotes, permits the diagnosis of fetuses expected to develop postnatal β-thalassemia major.