The aim of the study was to compare the levels of angiogenic markers and markers of placentation between pregnancies conceived with fresh (ET) and vitrified-warmed blastocyst transfer (FET). Methods Women with singleton pregnancies resulting from fresh ET or FET during the period between 2013 and 2017 were included in this prospective observational study. Fresh ET was performed in a stimulated and FET in natural cycle. At 6-7 weeks of gestation, after ultrasound confirmation of a single gestational sac with a viable embryo, serum levels of free β-hCG, pregnancy-associated plasma protein A (PAPP-A), placental growth factor (PIGF) and fms-like tyrosine kinase (sFlt-1) were measured. Data on the patients' characteristics, pregnancy complications and outcomes were collected from a questionnaire and National Perinatal Information System of Slovenia. Results Among 211 pregnancies, 126 were achieved with fresh ET and 85 with FET. There were no significant differences in perinatal outcome, pregnancy complication and PIGF level between the fresh ET and FET group. Women achieving pregnancy with FET had significant higher levels of free β-hCG (40.20 ± 30.62 IU/L vs. 28.74 ± 23.52, p = 0.002), PAPP-A (0.09 ± 0.06 vs. 0.06 ± 0.05 IU/L, p = 0.004) and sFlt-1 (596.19 ± 283.06 vs. 436.53 ± 248.23 pg/L, p < 0.0001) compared to women having conceived with fresh ET. There were no significant differences in the levels of evaluated biomarkers between patients with different pregnancy outcomes and complications. Conclusion Levels of angiogenic markers and markers of placentation differ between pregnancies achieved with fresh ET and FET which may reflect altered implantation and early placentation with some forms of assisted reproductive technologies.