Our objective was to compare luteal regression and pregnancies per AI (P/AI) after timed AI (TAI) for dairy cows receiving the first GnRH injection (G1) of an Ovsynch protocol in a low (<1.0 ng/mL) or a high (≥1.0 ng/mL) progesterone (P4) environment. Lactating Holstein cows (n = 851) at first or second and greater AI service were randomized to receive an Ovsynch protocol (G1; 7d, PGF_2α; 56h, GnRH; 16h, TAI) with 1PGF_2α (PGF) treatment (1PG; n = 440) or a modified Ovsynch protocol (G1; 7 d, PGF_2α; 24 h, PGF_2α; 32 h, GnRH; 16 h, TAI) with 2PGF treatments (2PG; n = 411). Blood was collected at G1, the first PGF treatment, and the last GnRH treatment (G2) of the Ovsynch protocol for analysis of P4. To eliminate cows undergoing early luteal regression, cows (n = 153) with P4 < 1.0 ng/mL at the first PGF treatment were excluded from the analysis. Cows with P4 < 0.4 ng/mL at G2 were defined as undergoing complete luteal regression after PGF, whereas cows with P4 ≥ 0.4 ng/mL at G2 were defined as failing to undergo complete luteal regression after PGF. Data were analyzed by logistic regression using PROC GLIMMIX of SAS. Overall, more (P < 0.01) cows with high P4 at G1 underwent luteal regression after PGF compared with cows with low P4 at G1 (92.2% vs. 82.8%, respectively). As expected, luteal regression after PGF was greater (P < 0.01) for 2PG (96.3%) compared with 1PG (83.8%) cows. For cows with low P4 at G1, more (P < 0.01) 2PG cows underwent luteal regression after PGF (69.6% vs. 96.2%, 1PG vs. 2PG). As a result, 2PG cows with low P4 at G1 had more (P < 0.01) P/AI compared with 1PG cows with low P4 at G1 (35.3% vs. 57.3% for 1PG vs. 2PG cows). By contrast, for cows with high P4 at G1, although luteal regression was greater for 2 PG (91% vs. 97%; 1PG vs. 2PG; P = 0.05), P/AI did not differ (35.9% vs. 39.7%; 1PG vs. 2PG; P = 0.39). We conclude that a second PGF injection 24 h after the first during an Ovsynch protocol increased luteal regression for cows with low or high P4 at G1 and increased P/AI for cows with low P4 at G1. Supported by USDA NIFA Hatch project 231440.