Endometrial mesenchymal stem cells (eMSCs) play a critical role in the pathogenesis of endometrium-related disorders, including recurrent implantation failure (RIF), due to their regenerative potential and regulatory influence on the endometrial microenvironment. Therefore, identifying eMSCsis helpful for understanding their role in endometrial repair, implantation, and disease pathology can drive advancements in clinical and biomedical research. The objective of the present study is to investigate the stem cell properties of endometrial cells isolated from patients with implantation failure following at least one euploid embryo transfer. This study involved sub-fertile women undergoing in vitro fertilization (IVF) with standard ovarian stimulation (antagonist or ultralong for endometriosis). A total of 23 women underwent ovarian stimulation, egg retrieval, fertilization, and had at least one euploid blastocyst planned for transfer. Embryo transfers, scheduled from October 2023 to April 2024, were performed at Paragon Fertility Bangkok and Srinakharinwirot University Hospital. The patients underwent mock endometrial preparation for further embryo transfer and scratching. Endometrial tissues were collected via sterile scratching, minced, and digested with 0.1% collagenase I. Cells were filtered, centrifuged, resuspended in DMEM/F-12 with supplements, seeded, and maintained at 37°C with 5% CO2. Cell characterization was performed using immunocytochemistry to confirm the expression of cytokeratin, vimentin, as well as CD44, CD90, STRO-1, and CD146. Pluripotent gene expression was analyzed by using quantitative real-time PCR. Self-renewal and clonogenic potential were assessed by CFU assay. A total of 4 out of 23 endometrial samples (17.4%) give rise to stem-like cells and enable continuous proliferation for up to passage 7. Of these, stromal stem cells, as confirmed by CD44, CD90, STRO-1, and CD146 positive immunostaining, were obtained from 2 donors. Gene expression analysis demonstrates that NANOG and OCT4 gene are highly and specifically expressed in stem cells. In addition, endometrial-derived cells exhibited clonogenic potential as demonstrated by CFU assay. In summary, stem-like cells with self-renewal and clonogenic potential were identified in a subset of endometrial samples from women with implantation failure.