Abstract Our objective was to determine whether a single examination of ovaries using transrectal ultrasonography at the first GnRH injection of a Presynch + Ovsynch protocol is a useful method for assessing cyclicity status and thereby enabling differential management of anovular vs. cyclic cows. Lactating Holstein cows (n=842) receiving a Presynch + Ovsynch protocol to initiate first postpartum timed artificial insemination (TAI) were used to compare 2 methods for assessing cyclicity status before TAI. For the standard method (using RIA), blood samples were collected at the second PGF2α injection of Presynch and the first GnRH injection of Ovsynch, and cows with serum progesterone ≥1.0ng/mL in one or both samples were classified as cycling, whereas cows with serum progesterone <1.0ng/mL in both samples were classified as anovular. For the practical method, transrectal ultrasonography (U/S) was used to determine the presence or absence of a corpus luteum (CL) at the first GnRH injection of Ovsynch, and cows without CL were classified as anovular, whereas cows with CL were classified as cycling. Statistical agreement (kappa) between the RIA and U/S methods to identify cycling cows was 0.66. Sensitivity, specificity, positive predictive value, and negative predictive value of U/S to identify anovular status were 85.7, 87.7, 64.7, and 95.9%, respectively. We conclude that assessing the presence or absence of CL at the first GnRH injection of a Presynch + Ovsynch protocol using U/S is a reliable and practical method for identifying the cyclicity status of cows before first TAI, but may slightly overestimate the proportion of anovular cows compared with the RIA method.