Introduction Duchenne muscular dystrophy (DMD) is one of the most common muscular dystrophy in childhood. The patients suffer progressive proximal muscle weakness and die from respiratory failure and cardiomyopathy in their 30’s. Pre-implantation genetic testing (PGT) is an alternative traditional prenatal diagnosis (PND). However, extremely large size of the gene and the diversity of mutations cause molecular genetic testing difficult and labor intensive. Modern haplotyping using aSNP and Karyomapping algorithm would be useful for diagnosis of monogenic disease. This study applied Karyomapping for PGT-M of DMD in 2 clinical PGT cycles in comparison to standard PCR testing techniques. Material and methods Two families at risk of having DMD offspring joined the project following thoroughly counselling and inform consent was obtained. The patients underwent IVF procedures. Embryo biopsy was performed on Day-5 post-fertilization and biopsied trophectoderm underwent whole genome amplification. SNP array with Karyomapping analysis was carried out for haplotyping as well as copy number variation (CNV). Multiplex PCR with mini-sequencing was performed alongside for confirmation standard molecular mutation analysis. Multiple microsatellites within dystrophin gene were also analyzed for linkage analysis and contamination identification. Results Nine embryos with good morphology from each patient were chosen for PGT. Karyomapping results of family TZ (DMD c.895G>T) revealed three normal, two carriers, two affected and two with intragenic recombination. Standard mutation analysis using multiplex fluorescent PCR incorporation with mini-sequencing and microsatellites analysis for linkage analysis confirmed haplotyping results in all embryos. In addition, karyomapping demonstrated one embryo with chromosome unbalanced, i.e. 46,XX, -6q (intragenic recombination). Therefore, three normal (two male and one female) and two carrier (both female) embryos with chromosomally balanced were fulfilled for transfer. During the first embryo transfer, one normal female embryo was chosen, no pregnancy was resulted. In the second transfer, one normal male embryo was transferred, one ongoing pregnancy was resulted. Karyomapping results of family JM (DMD exon 8-9 duplication) revealed two normal, two carriers, two affected and one with intragenic recombination. Standard microsatellites analysis for linkage analysis confirmed haplotyping results in all embryos. Additionally, karyomapping demonstrated one embryo with chromosome unbalanced, i.e. 45,XX, +2P,-22 (normal) and one embryo with uniparental disomy (UPD), i.e. 46,XX (normal). Therefore, two normal (both female) and two carrier (both female) embryos with chromosomally balanced embryos were fulfilled for transfer. All are being frozen waiting for transfer. Polymorphic marker analysis revealed the absence of extraneous DNA contamination. Conclusions Two clinical PGT-M cycles using karyomapping were performed for two families at risk of having DMD offspring. This study exhibits that aSNP provides the benefit of extra information of chromosome balance and parental origins, i.e. uniparental disomy in one of the embryo. Therefore, kryomapping can omit the risk of transfer chromosomally unbalanced embryos, termination of abnormal chromosome pregnancy later and the birth of abnormal chromosome babies. Therefore, karyomapping provides an accurate, quick, time saving for protocol development, universal PGT-M method for every monogenic disease of various types of mutations and also the advantage of CNV and parental origin information which is common abnormalities in pre-implantation embryos.