The inherited disorders of hemoglobin synthesis constitute themost commonmonogenic diseases worldwide. The clinical severity of β-thalassemia major and the sickle cell syndromes targets themas priority genetic diseases for prevention programs,which incorpo- rates population screening to identify heterozygotes,with the option of prenatal diagnosis for carrier couples. Rapid genotype characterization is fundamental in the diagnostic laboratory, especially when offering prenatal diagnosis. The application of real-time polymerase chain reaction (PCR) provides a means for rapid and potentially high-throughput assays, without compromising accuracy. It has several advantages over endpoint PCR analysis, including the elimination of post-PCR processing steps and a wide dynamic range of detection with a high degree of sensitivity. Although there are >180 mutations associated with the β- thalassemia and sickle cell syndromes, the relatively small size of the β-globin gene (<2,000 base pairs) and the proximity of most mutations facilitates the design of a minimal number of real-time PCR assays by using the LightCycler system (Roche Diagnostics [Hellas] A.E., Athens, Greece), which are capable of detecting the majority of most common β-gene mutations worldwide. These assays are highly appropriate for rapid genotyping of parental and fetal DNA samples with respect to β-thalassemia and sickle cell syndromes.