BACKGROUND Preimplantation Genetic Diagnosis (PGD) is presently a valid alternative for couples at high risk of pregnancies with genetic anomalies. Previously, unwielding and labour intensive methods such as restriction digestion, Denaturing Gradient Gel Electrophoresis or Heteroduplex analysis had to be used for mutation screening in PGD analysis. In order to overcome some of these limitations, we have adapted a mutation analysis method, known as Minisequencing, to PGD of different genetic diseases such as Cystic Fibrosis, -Thalassemia, Sickle Cell Anemia, Haemophilia A, Retinoblastoma, and Spinal Muscular Atrophy (SMA). In order to evaluate the reliability of this method, 887 blastomeres from 55 PGD cases were analysed simultaneously with both sequence analysis and with the minisequencing method. MATERIALS AND METHODS Polymerase Chain Reaction (PCR) strategy consisted in an initial multiplex external amplification followed by nested PCR. Reaction products were than analyzed both by sequence analysis and minisequencing methods, followed by capillary electrophoresis on automatic DNA sequencer. RESULTS AND CONCLUSIONS Minisequencing has provided interpretable results in 96.5% (856/887) of the blastomeres investigated, with respect to 86.7% (769/887) obtained with sequence analysis (P<0.001). Fifteen clinical pregnancies (15/55, 27%) have been generated; all the PGD results have been confirmed by prenatal diagnosis and 7 healthy babies were already born. Minisequencing has proved to be a useful method in PGD analysis, due to its elevated sensitivity, automation, and easy data interpretation.