INTRODUCTION Retinoblastoma is a malignant childhood tumor of the eye. Mutations in both alleles of the RB1 gene are prerequisite for the development of this neoplasm. In about 40% of patients, one of the mutant alleles is present in germ line cells and thus can be transmitted to offsprings. Molecular genetic testing of the RB1 gene is used for risk prediction in relatives. In the past years, Preimplantation Genetic Diagnosis (PGD) has become a valid alternative for couples at high risk of pregnancies with this kind of genetic anomalie. The intrinsic difficulties of this diagnostic approach require the use of extremely sensitive and automatized techniques that provide unambiguous and reliable results. Mutation analysis by means of sequencing of the genetic regions of interest is, at present, a better method of genetic analysis. While this technique guarantees the maximum interpretative trustworthiness its application in PGD analysis is time expensive and requires experience for data analysis. In order to overcome some of these limitations, we have applied a new method of genetic analysis, known as "Minisequencing", to a PGD case of Retinoblastoma. MATERIALS AND METHODS A couple who had an affected child for Retinoblastoma was referred to our center. The woman was also affected by the same disease. In order to find the mutation involved in this family, RB1 gene was completely sequenced. DNA samples of the family were isolated from peripheral bloods. RB1 gene, including all the exons, was amplified by polymerase chain reaction (PCR) and sequenced by automated DNA sequencing. For PGD analysis, PCR strategy consisted in an initial multiplex external amplification followed by nested PCR. PCR products were than analyzed both by sequencing and “minisequencing” methods, followed by capillary electrophoresis on automatic DNA sequencer. RESULTS AND CONCLUSIONS RB1 mutation analisys of affected patients revealed a new mutation as 160797insT. Screening of 18 blastomeres for the above mutation, carried out simultaneously with both traditional automated sequence analysis and “minisequencing”, evidenced perfectly overlapping data. “Minisequencing” has proved to be a very useful method in PGD analysis, due to its elevated sensitivity, automation, and easy data interpretation.. Our results indicate that “minisequencing” is a reliable, sensitive, efficient and easy-to-approach mutation analysis method, applicable to PGD on a single cell as well as to molecular diagnosis of known mutations. This represents the first italian PGD case of retinoblastoma.