Abstract: Objective To develop a rapid preimplantation genetic diagnosis method for α-thalassemia SEA deletion based on blastocyst cell whole genome amplification (WGA) combined with short fragment Gap-PCR. Methods Using multiple displacement amplification (MDA) WGA technique, we established a double-fluorescent PCR system of the housekeeping genes GAPDH and β-actin for WGA quality testing, and a genotyping PCR system of mutant and normal short sequences for α-thalassemia SEA deletion. The sensitivity and accuracy of this method for diagnosis of α-thalassemia SEA deletion were evaluated by detecting lymphocyte samples containing different cell numbers from carriers of SEA deletion. The applicability of this method was evaluated by testing of 12 blastocyst biopsy samples. Results Detection of lymphocyte samples with different cell numbers using the method developed in this study revealed no ADO in 3-cell samples, and the product quantity of WGA became stable for 4-cell samples. Genotyping of the 10 blastocyst biopsy samples with successful WGA showed a genotype of --SEA/αα in 5 samples and αα/αα in the other 5 samples, which were consistent with the verification results. Conclusion The method developed in this study is a complete testing process for 4-6 blastocyst biopsy cells to allow rapid, accurate, and cost-effective PGD genotyping of α-thalassemia SEA deletion using short fragment gap-PCR.