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Objective: To evaluate the impact of prolonged post-thaw embryos culture on pregnancy outcome during frozen embryo transfer cycles. Methods:This prospective cohort study evaluated 324 thaw transfer cycles with 819 embryos from 269 patients at the Center for Reproductive Endocrinology and Infertility of Hue University Hospital in Vietnam. These frozen embryo transfer cycles were divided into two groups at the time of thawing: the short culture group (2-hour post-thaw culture) and the ovight culture group (ovight culture for 18 h) before the embryo was transferred into the uterus. The rates of embryo intact, grade A embryo at frozen and transfer time and continuing cleavage were recorded. The clinical outcomes including serum beta-human chorionic gonadotropin, clinical pregnancy and implantation rate were evaluated after 14 days, 4 weeks, 6 weeks, respectively, after embryo transfer. Results: Human chorionic gonadotropin positive occurred in 39.5％ of patients in the short culture group compared to 25.9％ in the ovight culture group with risk difference (RD)=13.6％, relative risk (RR)=1.343, 95％ confidence interval (CI) 1.085-1.663,P<0.01. Clinical pregnancy rate of the short culture group and ovight culture group was 33.3％ and 24.1％, respectively (RD=9.2％,RR=1.242, 95％CI 0.996-1.549,P=0.06) and the implantation rate in the short culture group and ovight culture group was 16.5％ and 11.0％, respectively (RD=5.5％,RR=1.244, 95％CI 1.046-1.479,P=0.01). In women of advanced age (≥35 years) and women who received 3 embryos, pregnancy outcomes were found to be significantly (P<0.05) higher in the short culture than in the ovight culture group. Conclusions: The prolonged post-thaw culture period does not increase pregnancy outcome in comparison with the short culture.