Recently, Preimplantation Genetic Diagnosis (PGD) has been considered for several indications beyond its original purpose, not only to test embryos for a genetic disease, but also to select embryos for a non-disease trait, such as specific Human Leukocyte Antigen (HLA) haplotypes, related to immune compatibility with an existing affected child in need of an hematopoetic stem cell transplant. We have optimized an indirect single-cell HLA typing protocol based on a multiplex fluorescent polymerase chain reaction (PCR) of short tandem repeat (STR) markers scattered throughout the HLA complex. The assay was clinically applied in 60 cycles (54 for -thalassemia, 1 for Wiscott-Aldrich syndrome, 2 for Diamond-Blackfan anemia and 3 for Acute Lymphoid Leukaemia) from 45 couples overall, involving the testing of 486 embryos in combination with a genetic disease and 44 embryos for HLA matching only. In 848/922 (92.0%) of blastomeres a successful amplification was achieved, obtaining a conclusive HLA matching diagnosis in 483/530 (91.1%) of the embryos tested. In total, 74 (15.3%) embryos revealed an HLA match with the affected siblings, 55 (11.4%) of which resulted unaffected and 46 (9.5%) have been transferred back to patients in 30 clinical cycles. Nine pregnancies were achieved, 5 healthy HLA matched children have been already delivered and cord blood stem cells were transplanted to 3 affected siblings, resulting in a successful hematopoietic reconstruction. Multiplex PCR of STR markers located in the HLA region has revealed a reliable diagnostic tool for indirect HLA matching evaluation in single cells.