Objectives were to evaluate pregnancy per AI (P/AI) and pattern of insemination of 2 resynchronization protocols to maximize insemination rate based on estrus detection in lactating dairy cows. Holstein cows (n = 1,929) from 3 herds were assigned randomly to 2 presynchronization (Presynch) protocols based on their ear tag number. At non-pregnancy diagnosis (day of enrollment [d 0]), odd-numbered cows (P7GPG; n = 967) received a Presynch treatment of PGF_2α (PGF) and were enrolled in a resynchronization protocol on d 7 (GnRH on d 7 and 17; PGF on d 14; timed AI [TAI] on d 17). Even-numbered cows (P7GGPG; n = 962) received a Presynch treatment of PGF on d 0 and GnRH on d 7, and were enrolled in a resynchronization protocol on d 14 (GnRH on d 14 and 24; PGF on d 21; TAI on d 24). Detected estrus based on tail paint removal was conducted once daily during the study period and cows detected in estrus were inseminated within 1 h of detected estrus. Cows not detected in estrus by d 7 (P7GPG) or d 14 (P7GGPG) received their assigned TAI treatment. Cows were examined by transrectal ultrasonography to determine pregnancy status 36 d after AI. Binary outcomes were analyzed by logistic regression. The rate at which cows were inseminated was analyzed using the Cox’s proportional hazard model. No treatment difference was detected for the percentage of cows inseminated based on detected estrus (72.2%). Presynch treatment (P < 0.01), TAI (P < 0.01), and the interaction between TAI and Presynch treatment (P = 0.02) affected P/AI. Cows inseminated in estrus were more likely to become pregnant than TAI cows (30.5 vs. 23.1%). No difference was detected in P/AI for cows inseminated at estrus (30.5%). In contrast, for cows receiving a TAI, P/AI was (P < 0.01) greater for P7GGPG than P7GPG cows (28.9 vs. 17.6%). Hazard of insemination was affected (P < 0.01) by treatment because P7GPG cows were inseminated sooner than P7GGPG cows (AHR = 1.70 [95% CI = 1.53 to 1.89]). Presynchronizing cows with GnRH delays AI but improves P/AI of cows submitted to a TAI protocol.