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Context. According to in vitro experiments, up to 75% of morphologically normal human embryos cannot spontaneously leave the zona pellucida (ZP). Molecular mechanisms of hatching are still not completely studied. Objective. The aim of the study was to identify genetic predictors of human blastocyst hatching success in assisted reproduction. Methods. Hatching success assessment was performed after 144–146 hours post fertilization. The mRNA expression levels of cathepsin V (CTSV), GATA binding protein 3 (GATA3) and human chorionic gonadotropin beta subunit 3, 5, 7 and 8 (CGB) genes were detected by quantitative polymerase chain reaction. Patient(s) The study included 83 blastocysts, which were donated by 28 couples. The hatching group included 36 blastocysts (43.4%), which underwent spontaneous hatching. The control group included 47 blastocysts (56.6%) that did not undergo spontaneous hatching. Intervention(s). There was no interventions. Main Outcome Measure(s) We measured the rate of blastocysts with the thickening of the ZP, stage of the blastocyst development by Gardner scale, CTSV, GATA3 and CGB genes expression. Result(s). The rate of blastocysts with the thickening of the ZP was 14.4% and it was similar in the groups (11.1% in the hatching group 17.0% in the control group, p=0.4479). The rate of blastocysts of the 5th stage of development was significantly higher in the hatching group, and blastocysts of the 2nd and 3rd stage of development were detected only in the control group. In 63.8% of cases, inner cell mass (ICM) was evaluated as grade A (28.9%) and B (34.9%). In an equal number of cases, trophectoderm (TE) was evaluated as grade A (33.7%), B (36.2%) and C (30.1%). Grade A TE was detected in 47.2% of embryos in the hatching group but only in 23.4% of embryos in the control group. Grade C TE was detected in 44.7% of control blastocysts and only in 11.1% in the hatching blastocysts (p = 0.0030). The blastocysts in the control group had lower expression for the CTSV (3.9 vs 4.4, p<0.0001), GATA3 (4.5 vs 4.7, p=0.0005) and CGB (1.3 vs 2.1, p=0.0004) genes. Conclusions. We did not find an association between the rate of hatching failure and ZP thickness. The high blastocysts quality of Gardner scale parameters had a positive effect on hatching success. The expression of all three genes was higher in the group of blastocysts capable of spontaneous hatching. The quality of the blastocysts also affects the efficacy of spontaneous hatching.