Vitrification is a highly efficient technique for the cryopreservation of the human embryo. The effect of delayed blastulation may be responsible for implantation failures and negatively affects in vitro fertilization (IVF) outcomes. The current literature displays discordant results; some studies have announced higher pregnancy rates after day 5 (D5) transfer compared with day 6 (D6) transfer, while others have shown equivalent outcomes. In the present study an investigation into the clinical implications of delayed blastulation (D5 versus D6) was carried out. We performed a retrospective study comparing clinical pregnancies and implantation rates following warmed single blastocyst transfer (WSBT). All patients coming for a programmed warmed transfer at Edinburgh Assisted Conception Programme, EFREC, Royal Infirmary of Edinburgh, were included in this study and divided in two groups according to the day of blastocyst vitrification: D5 (n = 1563) and D6 (n = 517). The overall survival rate was 95.0% (1976/2080) with no significant difference between the D5 and D6 groups: 95.3% (1489/1563) and 94.2% (487/517) respectively. WSBT of D6 blastocysts resulted in a lower implantation and clinical pregnancy compared with D5 embryos. The implantation rate (IPR) and clinical pregnancy rate (CPR) were respectively 49.4% and 42.6% for the D5 and 37.4% and 32.2% for the D6 embryos, which was statistically significant. The multiple pregnancy rate was 1.32% (1.14% for D5 vs 1.84% for D6). Although the transfer of D6 vitrified-warmed blastocyst remains a reasonable option, priority to a D5 embryo would reduce the time to successful pregnancy.