Preimplantation diagnosis for X-linked disorders has been performed predominantly by gender determination, which, however, leads to the discarding of 50% unaffected male embryos. In an attempt to identify X-linked mutation-free embryos for transfer, the present authors introduced preimplantation genetic diagnosis (PGD), using a sequential first and second polar body analysis, as an alternative to gender determination. This method was offered to eight couples at risk for having children with X-linked disorders, including haemophilia B, fragile-X syndrome (FMR1), myotubular myotonic dystrophy (MTMD), ornithine transcarbamylase (OTC) deficiency and X-linked hydrocephalus. The first and second polar bodies were removed following maturation and fertilization of oocytes in a standard IVF protocol and analysed using a multiplex nested polymerase chain reaction (PCR), involving testing for mutations simultaneously with linked markers. Overall, 13 PGD cycles were performed, resulting in the detection of 25 embryos with the predicted mutation-free maternal contribution; these embryos were transferred back to the patients in all cycles, yielding four clinical pregnancies. Four children were born following these pregnancies, including three unaffected and one with misdiagnosis as a result of allele dropout (ADO), which was predictable in the case of FMR1. Presented results demonstrate the clinical usefulness of the specific polar body testing for X-linked disorders as an alternative to PGD by gender determination.