Objective To assess the efficacy and clinical outcome of PGT-M undertaken on Day-3, Day-4 and Day-4 “delayed” embryos that were unsuitable for biopsy on Day-3. Design and setting Cohort-historical study of all consecutive patients admitted to the IVF-PGT-M program in a large tertiary center. Main outcome measure(s) The pregnancy rates and the percentages of complete, incomplete diagnosis, PCR failure, abnormal embryos in PGT of Day-3 cleavage-stage, Day-4 and Day-4 “delayed” embryos. Patients and methods We reviewed the medical files of all consecutive patients admitted to our IVF for a fresh IVF-PGT-M cycle. Patients were divided into 3 groups according to the day of blastomere biopsy: Day 3 cleavage-stage, Day-4 morula and Day-4 “delayed” embryos. The laboratory data, genetic diagnostic and clinical results were collected and compared between the different study groups. Results Nine hundred and six patients underwent PGT-M cycles in our PGT program: 747, 127 and 32 in the Day-3, Day- 4 and Day-4 “delayed” groups, respectively. Ongoing pregnancy rates per transfer and per patient (15.8% and 9.4%, respectively) were non-significantly lower in the Day-4 “delayed”, compared to Day-3 (21.4% and 17.5%, respectively) and Day-4 (24.3% and 19.7%, respectively). When comparing ALL morulas (Day-4 and Day-4 “delayed”) to ALL cleavage-stage embryos (Day-3, Day-4 and Day-4 “delayed”), a significantly higher ongoing pregnancy rate was demonstrated following the transfer of embryos derived from morula biopsy, as compared to biopsy at the cleavage-stage (33.3% vs 20.5%, p<0.03, respectively). Conclusion Day-4 embryo biopsy is feasible and yields comparable and even higher ongoing pregnancy rate if undertaken at the morula stage. Further studies evaluating the cumulative live-birth rate per started cycles in Day-3 vs Day-4 embryo biopsy for PGT-M are warranted.