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Graphical Abstract Summary: This study evaluated the timing of artificial insemination (AI) with frozen-thawed sex-sorted semen on pregnancy per AI in dairy heifers. A 6-day progesterone Co-Synch protocol was used for ovulation synchronization of 823 dairy heifers, with timed AI (TAI) coincident with (TAI-0) or 8 hours (TAI-8) after the second injection of gonadotropin-releasing hormone (GnRH), corresponding to either 48 or 56 hours after removal of the progesterone-releasing intravaginal device. We hypothesized that TAI-8 heifers would have greater pregnancy rates than TAI-0 heifers by delivering viable sex-sorted semen to the uterus closer to the time of ovulation. The study demonstrated that delaying the normal timing of AI of dairy heifers with frozenthawed sex-sorted semen by 8 hours, thereby delivering sperm cells closer to the presumed time of ovulation, improved pregnancy per AI.