© 2022 Wiley-VCH GmbH.The objectives of the study were to determine (1) oestrous expression rate and (2) the effect of oestrous expression prior to progesterone-based Ovsynch protocol on pregnancy rate in Holstein cows. All cows (n = 917) were subjected to 7-day progesterone-based Ovsynch protocol. In this protocol, cows that expressed oestrus before (HEAT1) the scheduled second GnRH were inseminated 20 h later after the onset of oestrus without GnRH administration. Cows that expressed oestrus after the second GnRH administration (HEAT2) or did not express oestrus (NOHEAT) received fixed-timed AI. Oestrous expression was determined by using activity-rumination monitoring system and all cows were inseminated with sexed semen. Oestrous expression rate prior to FTAI was 40.5% and the majority (p <.01) of oestrous expression were in HEAT2 compared with HEAT1 in both primiparous (71.8 vs. 28.1%) and multiparous cows (69.5 vs. 30.5%). The mean interval from intravaginal device removal to the onset of oestrus was 47.4 ± 0.9 h and 62.9 ± 0.5 in HEAT1 and HEAT2, respectively. Primiparous cows (47.7%) had a higher (p <.01) expression rate compared with multiparous cows (37.2%). Overall pregnancy rate was 37.4% and there was two-way significant interaction between parity and oestrous expression on pregnancy rate (p <.01). Both primiparous (48.1 vs. 35.8%) and multiparous cows (47.4 vs. 28.4%) that expressed oestrus had greater (p <.01) pregnancy rate compared with cows that did not express oestrus. There was no difference in pregnancy rates of HEAT1 and HEAT2 in both primiparous (44.7 vs. 49.5%) and multiparous cows (47.2 vs. 47.6%). Pregnancy rate was not influenced (p =.21) by milk production (high or low) in both primiparous (47.6 vs. 48.6%) and multiparous (54.9 vs. 42.1%) cows that expressed oestrus, respectively. In conclusion, cows showing oestrus before or after second GnRH of the Ovsynch protocol had greater pregnancy rate than cows not showing oestrus.