Our objective was to assess the association between progesterone (P4) concentration at each treatment of an Ovsynch protocol and pregnancies per AI (P/AI) to timed AI (TAI) in lactating Holstein cows. Data from 7,792 cows from 14 experiments in which P4 was measured during an Ovsynch protocol [GnRH (G1); 7 d PGF_2α (PGF); 56 h, GnRH (G2); 16 h, TAI] were analyzed. Cows receiving exogenous P4 or that received 2 injections of PGF or an increased dose of PGF were excluded from the analysis. The association between P4 during the Ovsynch protocol and P/AI to TAI was analyzed independently because P4 was not measured for all cows at every time point in all experiments. Data were analyzed by logistic regression using the GLIMMIX procedure of SAS. At G1, cows (n = 6,144) were stratified into 9 P4 categories from 0 to ≥7 ng/mL using 0.5 ng/mL increments. Overall, P/AI differed (P < 0.01) among P4 categories at G1 with fewer P/AI for cows with P4 < 0.5 ng/mL (30%; 375/1,259) or P4 > 7.0 ng/mL (30%; 115/381) compared with cows with intermediate P4 (41%; 1,839/4,504; calculated relative decrease in P/AI of 27%; 41–30 = 11/41 = 27%). At PGF, cows (n = 3,383) were stratified into 9 P4 categories from 0 to ≥8 ng/mL using 1.0 ng/mL increments. Overall, P/AI differed (P < 0.01) among P4 categories at PGF with a 51% relative decrease in P/AI for cows with P4 < 1.0 ng/mL (21%; 140/681) compared with cows with P4 > 1.0 ng/mL (43%; 1,151/2,702). At G2, cows (n = 3,148) were stratified into 8 P4 categories from 0 to ≥0.7 ng/mL using 0.1 ng/mL increments. Overall, P/AI differed (P < 0.01) among P4 categories at G2 with a 66% relative decrease in P/AI for cows with P4 > 0.4 ng/mL (14%; 61/435) compared with cows with P4 < 0.4 ng/mL (41%; 1,125/2,713). Based on this large data set, suboptimal P4 concentrations could be identified at G1 in 26% of cows (26% lower P/AI), at PGF in 21% of cows (51% lower P/AI), and at G2 in 14% of cows (66% lower P/AI). We conclude that achieving optimal P4 during an Ovsynch protocol may allow for a dramatic increase in fertility in lactating dairy cows. Supported by USDA NIFA Hatch project 23144.