A study evaluated the effect of a 16-h delayed AI following GnRH administration in a hybrid estrus detection and time AI protocol in heifers not in estrus at the time of scheduled AI. Angus-based, crossbred heifers (n = 453) of the same origin were managed at the Kelly Ranch (Sutherland, NE) or the University of Nebraska West Central Research and Extension Center (North Platte, NE). Estrus was synchronized utilizing the melengestrol acetate (MGA)-PG protocol; heifers received MGA for 14 d. Nineteen d later, on d 33 of the protocol, heifers received a PG injection and estrus detection aids (Estrotect) were applied. Heifers were considered to have expressed estrus when greater than 50% of the rub off coating was removed from the Estrotect. Heifers (n = 319) were then removed from the herd and AI 12 h later (ESTRUS). Seventy-two hours following the PG injection, heifers whose Estrotect were less than 50% activated were administered GnRH, and randomly assigned to 1 of 2 treatment groups; 1) immediately AI (n = 70, GNRH1) or 2) AI 16 ± 1 h following GnRH injection (n = 64, GNRH2). Data were analyzed using the GLIMMIX procedure of SAS (SAS Inst. Inc., Cary, NC) and the proportion of pregnant heifers was found using an odds ratio utilizing the ILINK function. Pre-breeding BW was similar (P = 0.58) between ESTRUS, GNRH1, and GNRH2 (351, 346, and 349 ± 6 kg, respectively). Furthermore, pregnancy diagnosis BW among the treatments was similar (P = 0.48; 376, 380, and 377 ± 6 kg; ESTRUS, GNRH1, and GNRH2, respectively). Heifers who were AI on their estrus, as determined by an activated Estrotect, had significantly higher (P < 0.01) pregnancy rate compared with heifers in both GNRH1 and GNRH2 groups (70 vs. 56, 47 ± 6%). Pregnancy rates did not differ (P = 0.56) between GNRH1 and GNRH2 (56 vs. 47 ± 6%). Heifers in all groups reached a similar (P = 0.59) percentage of mature BW before the breeding season (63 ± 1%). Final pregnancy rate was not different (P = 0.54) for ESTRUS, GNRH1, and GNRH2 heifers (92, 89, 91 ± 4%). There was no significant benefit to delayed AI of non-estrus beef heifers compared with traditional timed AI at the time of GnRH injection.