The objectives of this observational study were to determine the optimal progesteronemia threshold for defining anovulation at 50 days in milk (DIM) in dairy cows and to identify risk factors for this condition. A total of 3,776 cows from 100 Holstein dairy herds were enrolled in this cow-level study. During farm visits, cows were bled at 1-14DIM to quantify ketonemia and glycemia, examined at 30-43 DIM to diagnose purulent vaginal discharge, cytological endometritis, and leukocyte esterase endometritis, and were bled at a 14d interval to quantify progesteronemia. Multiple progesteronemia thresholds were tested to identify the one providing the highest sum of sensitivity and specificity to predict the pregnancy status at first service. The optimal threshold found for defining anovulation at 50DIM was ≤0.90 ng/mL. The final model for risk factors included parity group, season of calving, cytological endometritis, hyperketonemia, hypoglycemia, and the two-way interaction term of hyperketonemia and hypoglycemia. Overall, these results suggest that a progesteronemia threshold of ≤0.9ng/mL could be used to define anovulation at 50 days in milk in dairy cows.