AbstractThe aim of the present study was to compare the endometrial gene expression of epidermal growth factor receptor (EGFR), nodal growth differentiation factor (NODAL), prostaglandin‐endoperoxide synthase 2 (PTGS2), oestrogen receptor 1 (ESR1) and progesterone receptor (PGR) in repeat breeder cows (RBC) and non‐RBC during diestrus. Endometrial samples were collected by cytobrush technique and stored in RNA stabilizing solution at −20°C until RT‐qPCR analysis. Differences in endometrial mRNA expression of selected genes were assessed by ANOVA and simple (r) and the partial correlations (rp) among selected genes were performed. Results demonstrated that mRNA expression of EGFR and NODAL were higher in RBC than in non‐RBC (3 and 25‐fold change, p < .01 and p < .01, respectively), while the mRNA expression of PTGS2 was lower (1.56‐fold change, p < .01). Although there were no differences detected in the mRNA expression of ESR1 and PGR, there was a positive correlation between the expression of ESR1 and EGFR (0.84, p < .05) and a negative correlation between PGR and PTGS2 (−0.49, p < .05). In conclusion, the difference on the endometrial mRNA expression of the genes included in the study between RBC and non‐RBC indicates a deregulation of important mechanisms that are vital to establish a successful pregnancy. Thus, the present study provides useful insight as a base for future studies to elucidate the causes of RBC.