Introduction The mitochondrial genome contains single nucleotide variants (SNVs) that can be used to differentiate individuals. Additionally, mitochondrial DNA (mtDNA) is maternally inherited, providing a novel opportunity for DNA-based confirmation of maternal origin of embryo biopsies during Preimplantation Genetic Testing for Aneuploidy (PGT-A) by Next Generation Sequencing (NGS). Typically biopsies from different patients are processed and sequenced together in parallel as high throughput technologies necessitate sample batching for economical purposes. The mitochondrial genome is sequenced during PGT-A and the depth and breadth of coverage obtained from DOPlify® and the PG-Seq™ kit readily allows SNV analysis, even from a 48 sample NGS run. Here we investigate the clinical performance of RHS’ custom Embryo ID Single Nucleotide Variant (SNV) Panel. Material and methods A large putative panel of mitochondrial SNVs was compiled from published literature and by comparative analysis of PG-Seq™ data in IGV. Any SNVs associated with disease- related markers or in regions of lower depth of coverage using DOPlify™ were excluded. A single panel comprising 48 SNVs was compiled for evaluation. PG-Seq™ typically analyses 48 embryo biopsies in a single NGS run. Therefore, we modelled three separate NGS run scenarios; 4 embryos per patient (ie 12 unique mtDNA genomes), 2 embryos per patient (ie 24 mtDNA genomes), or 1 embryo per patient (ie 48 different mtDNA genomes) and tested performance of the panel in each scenario. For each scenario the appropriate number of mtDNA genomes were randomly selected from a publicly available dataset comprising 377 mtDNA genomes representing the global population (mtDB; http://www.mtdb.igp.uu.se/ ), and the panel's ability to differentiate the samples was assessed. This was replicated 10,000 times for each scenario. Results In the 10,000 simulated cases using the 377 mtDNA genome database and modelling embryos from 12 patients, on average, the SNV panel differentiated 91.5% of embryos. In the simulated cases with embryos from 24 patients, on average, the SNV panel differentiated 84.6% of embryos. In simulated cases with embryos from 48 patients, on average, the SNV panel differentiated 75.0% of embryos. In a clinical setting, if embryo signatures match, the rest of the mtDNA genome can still be used to further differentiate samples. It should be noted that maternally-related patients will share mtDNA genomes. In this case, it will not be possible to distinguish between embryos from these patients using the mitochondrial genome. Conclusions RHS’ PG-Seq™ and the Embryo ID Single Nucleotide Variant (SNV) Panel readily generates a unique embryo signature providing a simple novel identification protocol. RHS’ Embryo ID panel has the potential to improve PGT-A practice by providing a DNA-based confirmation of maternal origin and sibling embryo identification. This could be used for sample tracking within an IVF or genetic service provider laboratory. The discriminatory power of the panel may be improved simply by inclusion of population-specific SNVs. Embryo ID will be incorporated into a future release of PG- Seq™ software.