Objective: This study aims to determine the impact of assisted hatching (AH) on pregnancy outcomes in vitrifiedwarmed blastocyst transfers, and evaluate if embryo expansion or morphology influences outcomes. Methods: A retrospective cohort study was performed including vitrified-warmed blastocyst transfers at our clinic between 2013 and 2017. Of the 2165 embryo transfers, 1986 underwent laser AH and 179 were non-assisted hatched (NAH). The primary outcome was live birth. Secondary outcomes included conception, implantation, clinical pregnancy, clinical pregnancy loss, and monozygotic twinning (MZT). Results: AH and NAH groups had similar rates of conception (38.7% vs 42.1%), implantation (26.2% vs 27.3%), clinical pregnancy (29.1% vs 30.3%), clinical pregnancy loss (24.0% vs 17.8%), live birth (19.9% vs 20.5%), and MZT (2.08% vs 2.86%). Five pairs of dichorionic/diamniotic twins resulted from single embryo transfers. AH of embryos with expansion grades ≤3 was associated with lower rates of conception (32.5% vs 44.3%%, p < 0.05) and clinical pregnancy (24.0% vs 32.8%, p < 0.05). Conclusion: AH prior to transfer of vitrified-warmed blastocysts was not associated with improved pregnancy outcomes. The identification of dichorionic/diamniotic twins from single blastocyst transfers challenges the previously held notion that dichorionic/diamniotic MZTs can only occur from division prior to the blastocyst stage. Prospective studies are needed to validate the novel finding of lower rates of conception and clinical pregnancy after AH in embryos with lower expansion grade.