Thalassemia and hemoglobinopathies are very high prevalence in Thailand, leading to major health and socio-economic problem of the country. The national prevention and control program of thalassemia has been implemented throughout the country for years. One objective of this program is to prevent birth of new case with severe thalassemia diseases including Hb Bart’s hydrops fetalis, homozygous b-thalassemia and Hb E/b-thalassemia. To succeed the policy, prenatal diagnosis is available for pregnancies at risk of severe thalassemia. However, the presence of maternal contamination in chorionic villus sampling or amniotic fluid samples poses a serious pre-analytical risk for prenatal misdiagnosis. It may lead to a false positive results based on the presence of mutation-positive maternal cells. This study aimed to determine the heterozygosity of APO B VNTR and the effectiveness of prenatal diagnosis when using APO B VNTR alone and combination with the routine marker, D1S80 VNTR. The study was conducted on left-over DNA from pregnant, husbands and fetal tissues (100 families) which came for prenatal diagnosis at Thalassemia unit, CMDL, AMS, Khon Kaen University. Prenatal diagnosis for severe thalassemia was performed using Gap-PCR for detection of Hb Bart’s hydrops fetalis and ASPCR for homozygous b-thalassemia and Hb E/b-thalassemia. These VNTR loci were analyzed using PCR method and subsequently 2% agarose gel electrophoresis. From prenatal diagnosis, fetuses with normal genotype, Hb Bart’s hydrops fetalis, homozygous b-thalassemia and Hb E/b-thalassemia were observed for 18, 13, 3 and 16%, respectively. The heterozygosity of D1S80 and APO B VNTR were 82.5 and 47.5%, respectively. The using of D1S80 and APO B VNTR provided informative data for 59 and 39%, respectively. The combination of two VNTRs provided information on 77% of cases which giving more informative data for 18%. The results of this study indicated that APO B VNTR has a relatively low heterozygosity, using APO B VNTR alone is not suitable for routine maternal contamination testing. However, using APO B VNTR in combination with D1S80 VNTR could give more informative and helpful in prenatal diagnosis of severe thalassemia.