Introduction: Mutation-directed PGD protocols are successfully used for mutation screening in the majority of PGD cases. For diseases involving a heterogeneous spectrum of mutations identified, the development of a mutation-based PGD strategy could be not practical because it requires time and resources for standardization of PCR protocols unique for the specific mutations. The use of a diagnostic strategy capable of detecting a wide spectrum of mutations and compound genotypes can be more feasible. We have applied a new method of genetic analysis, called "Minisequencing", to PGD of monogenic disorders. The main benefit of the minisequencing strategy is the use of a mutation analysis protocol based on a common procedure, irrespective of the mutations involved. We analyzed PCR products from 962 blastomeres including 62 PGD cycles of different genetic diseases, such as Cystic Fibrosis, -Thalassemia, Sickle Cell Anemia, Haemophilia A and B, Retinoblastoma, Spinal Muscular Atrophy, and Lesch-Nyhan syndrome. Material and methods: PCR amplification was performed on 1052 blastomeres biopsied from 551 embryos. The PCR strategy consisted in an initial multiplex external amplification followed by nested PCR, specific for each region involving mutations. Mutation analysis was carried using two different techniques simultaneously: sequence analysis and minisequencing. Results:. We obtained positive amplification in 962 blastomeres. The overall amplification rate was 91.4% and the average ADO rate was 10.1%. Interpretable results were obtained in 96.9% (932/962) of the blastomeres analysed with minisequencing, with respect to 87.2% (839/962) of blastomeres investigated with sequence analysis. After the clinical cycles, blastomeres from 147 non-transferred embryos were reanalyzed to verify the diagnosis, and the genetic status was confirmed in all PGD cases. Sixteen clinical pregnancies resulted from these PGD cases; conventional prenatal diagnosis confirmed all the PGD results, and 12 healthy babies have already been born. Conclusions: Minisequencing has proved to be a very useful technique, simplifying single cell DNA analysis. It has been shown to be a reliable and generally applicable alternative for mutation-directed PGD protocols. Its applicability in PGD could be helpful, especially in cases where the mutation(s) involved is difficult to assess by restriction analysis or other commonly used methods.