beta-thalassemia is one of the most common inherited diseases with incidence varying between 3% and 10% in the high-prevalence regions of South China. The molecular defects are mostly due to single-nucleotide substitutions, minor insertions, and deletions in the beta-globin gene. Large-scale population genetic screening combined with prenatal diagnosis is necessary for the effective prevention of this disease. We present a single base extension (SBE) method based on homogenous fluorescence polarization (FP) for simultaneous detection of the eight most common causative mutations [CDs 41-42 (-TCTT), IVS-2-654 (C-->T), -28 (A-->G), CD17 (A-->T), CD 71/72 (+A), CD26 (G-->A), -29 (A-->G), and CD43 (G-->T)] in the beta-globin gene in a Chinese population. This assay has been validated by a blind experiment with 100 clinical samples previously characterized by reverse dot-blot and direct sequencing. The results demonstrate that this high-throughput method is simple, reliable, and cost effective. We expect this approach can be used in large-scale genetic screening for beta-thalassemia.