Hemophilia A is a common X-linked bleeding disorder caused by mutations in the coagulation factor VIII gene. The entire coding and essential sequences of the factor VIII gene were generated by a combination of genomic DNA amplification and long reverse transcription-polymerase chain reaction (long RT-PCR) using factor VIII transcripts prepared from lymphocytes. Mutations were then screened by non-radioactive single strand conformation polymorphism (SSCP) analysis and characterized by DNA sequencing. We have identified six potentially pathogenic mutations in the factor VIII gene in Thai hemophilia A patients, including two nonsense mutations (R-5X and R1966X), three missense mutations (D542Y, G1850V, and G2325C), and a 4-bp insertion (ACTA) at codon 2245. Three of these mutations (D542Y, G2325C, and 4-bp insertion) have never been previously reported, and the ins2245 is the first example of such insertion probably causing factor VIII elongation. R1966X, D542Y, G1850V, and 4-bp insertion were associated with a severe hemophiliac phenotype whereas R-5X and G2325C were observed in moderately affected patients. Mutations in the factor VIII gene in Thai hemophilia A patients are likely to be heterogeneous. This study represents the first attempt to further the understanding of the molecular basis of hemophilia A in Thai.