OBJECTIVE: Pregnancy in cattle after embryo transfer (ET) is influenced by several factors, including embryo quality. Therefore, preparing high-quality embryos with the greatest developmental potential is essential for achieving a successful pregnancy after ET. Meanwhile, blastocysts produced by in vitro fertilization (IVF) procedure have different developmental speed during in vitro culture (IVC) and they exhibited different competence in the establishment of pregnancy. METHODS: This study aimed to identify the comparative features of early-, mid-, and late-developing bovine IVF blastocysts, when they first appeared at Day 7, 8, and 9 during IVC, respectively. In addition, the correlations between their molecular features and pregnancy ability were analyzed. RESULTS: The results showed no difference in the morphological characteristics, including total cell count and diameter, between the Day 7, 8, and 9 blastocysts. However, the pregnancy rate post-ET was significantly different between the groups at 51.7%, 36.7%, and 17.8% for Day 7, 8, and 9 blastocysts, respectively. During early embryo development, late-developing blastocysts demonstrated a reduced cell count in the inner cell mass and decreased expression of the early embryo developmental genes (Oct4 and Sox2) compared with the early- and mid-developing blastocysts. In addition, the number of apoptotic cells and apoptosis-related gene expression (increased Bax and decreased Bcl2) gradually elevated from the Day 7 to Day 9 blastocysts. However, there was no difference in mitochondrial activity and mitochondria-relevant gene expression (Tfam and Cox1) between the groups. Correlation analysis identified a significantly negative correlation between the pregnancy rate and the blastocysts' degree of apoptosis, indicating that the low pregnancy ability of late-developing blastocysts was mainly caused by increased apoptosis. CONCLUSION: This study's results may contribute to the field of animal biotechnology by assisting in establishing an improved strategy for bovine ET with IVF embryos.