To assess whether there is an association between extended in vitro culture based on embryo developmental stage at transfer and pre-malignant gestational trophoblastic disease (GTD) risk of molar pregnancy during assisted reproduction. A retrospective study was carried out using Human Fertilization and Embryology Authority (HFEA) anonymized register from 1999 to 2016. A total of 540,376 cycles were eligible to be included in the study after excluding any kind of donor treatment or surrogacy, frozen embryo transfers, and cycles with incomplete data. Subgroup analysis was carried out in subjects with primary infertility aiming to exclude an increased risk in those with a previous GTD. Multivariate logistic regression analysis was used to adjust for possible confounders, and the effect of day of embryo transfer in IVF (in vitro fertilization)/ICSI (intracytoplasmic sperm injection) treatment on a molar pregnancy GTD outcome was analyzed. The prevalence of a molar pregnancy GTD among the study population was 3.4/10,000 livebirths (53/156,683) with a higher risk in the over 40 age category. No significant difference of pre-malignant GTD incidence was seen between IVF and ICSI (0.01% vs 0.009% respectively). No association was seen with GTD based on type/cause of infertility or number of embryos transferred. Crude (1.06; 95% CI 0.852-1.31) and adjusted (1.07; 95% CI (0.857-1.32) odds ratios were calculated to see an association between day of embryo transfer and the occurrence of a GTD. There was no association between day of embryo transfer and molar GTD risk after adjusting for age and secondary infertility. No significant association between pre-malignant molar gestational trophoblastic disease and extended in vitro embryo culture was found after analyzing 540,376 cycles of IVF and ICSI.