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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. [ABSTRACT FROM AUTHOR]